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THE BIOLOGY OF

AND OF SOME OTHER
COELENTERATES;

EDITED BY: HOWARD M. LENHOFF
V. FARNSWORTH LOOMIS

-gSu^

THE BIOLOGY OF

HYDRA

AND OF SOME OTHER
COELENTERATES:

1961

EDITED BY: HOWARD M. LENHOFF
W. FARNSWORTH LOOMIS

THE BIOLOGY OF

HYDRA

AND OF SOME OTHER
COELENTERATES :

1961

Edited by

Howard M. Lenhoff

and

W. Farnsworth Loomis

UNIVERSITY OF MIAMI PRESS
CORAL GABLES, FLORIDA

l)y

The Loomis Institute tor Seientifie Keseareli, Iiie.

Library of Congress Card Number 61-18157

Printed in the United States of America

bj-

Rose Printing Company

Tallahassee, Miami, Jacksonville, St. Augustine

Forevv^ord

"Further, I discovered a little animal whose body was at times
long, at times drawn up short, and to the middle of whose body ....
a still lesser animalcule of the same make seemed to be fixed fast
by its hinder end .... [At that time the little animalcule] had only
four very short little horns, yet after the lapse of sixteen hours I
saw that its body and its horns had increased in bigness, and four
hours later still I saw it had forsaken its mother.''^

In this remarkable letter written on Christmas Day, 1702, An-
tony van Leeuwenhoek amazed members of the Royal Society by
annonncing a discovery of dual significance. While reporting the
initial description of the organism which we now call hydra," he
also described the first instance of asexual reproduction ever ob-
served in animals. Thus, from their very discovery hydra have
served to reveal new biological phenomena.

More startling findings with hydra followed when, in 1744,
Abraham Trembley published in his superb Memoires an exposition
of: the first controlled experiments on regeneration; the first suc-
cessful animal grafting experiments; the first investigations of photo-
taxis in lower invertebrates; the first vital staining of tissues; and
thorough proof of asexual reproduction by budding. Two centuries
have passed since Trembley made these revolutionary discoveries,
an interim during which research on hydra was sporadic, and hydra
were relegated to a subsidiary role in classroom instruction. In the

1 Letter 149, December 25, 1702. Quoted in Antony van Leeuwenhoek and his
"Little Animals" by Clifford Dobell, Dover Pubis., N. Y., 1960, pp. 280-281.

- In this volume we have adopted, whenever possible, the following usages for
purposes of unifonnity and clarity: (a) Hydra, when referring to one or more speci-
mens of this genus if the species has already been clearly indicated; (b) hydra,
when referring to one or more specimens of the Hydridae in general, and when the
species is not indicated; (c) hydras, when referring to a number of genera of the
Hydridae.

last decade, however, a renaissance in the use of hydra as a labora-
tory animal has been in progress.

The return of hydra to their original status as laboratory ani-
mals is marked by the pul^lication of Tlie Biolog,y of Hydra : 1961.
This is the first book since Trembley's Memoires devoted to original
research reports dealing for the most part with hydra. The present
volume is a record of a symposium on the Physiology and Ultra-
structure of Hydra and of some other Coelenterates held March 29-
31, 1961, at the Fairchild Tropical Gardens, Coral Gables, Florida.

In this symposium. North American workers representing many
different fields of biology described their current work. They started
with a discussion of the fine structure of hydra cells and mesoglea.
Following a session devoted to the development, chemistry, and
function of nematocysts, they considered the sul^jects of feeding
and nutrition. Next, research on tissue culture, symbiosis, and cal-
cification were discussed. A session concerning the various forces
responsible for the patterns of colonial hydroids led, in turn, to a
consideration of cellular differentiation and then of aging in both
mortal and immortal coelenterate types. Appropriately, attention
turned at last to regeneration and to new birth as seen in budding.

In organizing this symposium, the editors desired to bring about
an integration of knowledge from a large variety of disciplines.
Electron microscopists, naturalists, biochemists, and developmental
biologists ordinarily do not read or publish in the same journals.
The aim of the symposium was, therefore, to effect an interdisci-
plinary synthesis which might otherwise take years by normal chan-
nels. Accordingly, the discussions that followed each talk are
included because they point out some of the many unsolved prob-
lems and therefore should prove of value in stimulating further
investigations.

Much of the work presented at this symposium is in an early
stage. At times we have thought that perhaps these results are
too preliminary and should only be compiled after more data have
been accumulated. The situation is analogous to constructing a new
building. At times we might feel that all such work should proceed
behind walls marked "Work in Progress. No Admittance." At other
times we are intrigued with the very smell of sawdust and of wet
paint. It is in this latter spirit that the volume was compiled, for

these efforts, given time, may well show that hydra are particularly
favorable material for the investigation of cellular and intercellular
problems. History at least supports this view, because it was in
hydroid material that asexual reproduction and regeneration were
first discovered over two hundred years ago.

'7 cut off the heads of the one that had seven, and after a few
days I saw in it a prodigy scarcehj inferior to the fahidous Hydre of
Lernaea. It acquired seven new heads ... .But here is something
more than the legend dared to invent: the seven heads that I cut off
from this Hydre, after being fed, became perfect animals. . . ."^

W. Farnsworth Loomis, M.D. Howard M. Lenhoff, Ph.D.

Greenwich, Connecticut Miami, Florida

September 21. 1961

^ Abraham Trembley, 1744. Memoircs, pour servir « Vhistoire d'un genre de
polypes d'eau douce, a bras en forme de comes. Leide (Verbeek), p. 246. Quoted
in Abraham Trembley of Geneva, John R. Baker, Arnold & Co., London, 1952, p. 34.
(A complete translation, to be published, S. G. Lenhoff and H. M. Lenhoff, University
of Miami Press. )

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Participants

Barbara O. Alving
Eric Alving
Reza Bashey
Robert J. Boucek

B. Bourne
John Bovaird
Patricia Broberg
Robert R. Bryden
Stanley Burg
Allison Burnett
F. Gray Butcher
Edward L. Chambers
George B. Chapman
David L. Claybrook
Sears Crowell
Robert E. Eakin
Don W. Favv^cett
Hector Fernandez
Bernard Fritzie
Chandler Fulton
Geraldine F. Gauthier
Lauren C. Gilman
Thomas Goreau

C. T. Grabom^ski
Cadet Hand
Arthur Hess
Ray M. Iverson
Edward Kline
A. R. Krall
Charles E. Lane
Edward Larson
W. Henry Leigh

Howard M. Lenhoff
Yu-YiNG Fu Li
Alfred L. Loomis
W. Farnsworth Loomis
Philip Lunger
Charles F. Lytle
G. O. Mackie
A. G. Matoltsy
J. Marsh
Edgar J. Martin
N. Mason

Leonard Muscatine
Nancy L. Noble
Edward E. Palincsar
Helen D. Park
L. M. Passano
John H. Phillips
Earl R. Rich
Gordon C. Ring
D. M. Ross
K. Savard
Harriet Schapiro
David B. Slautterback
Daryl Stafford
Bernard L. Strehler
W. J. VAN Wagtendonk
Stephen A. Wainwright
Peter Wangersky
Eleanor D. Wangersky
John H. Welsh
Richard L. Wood
Edmund Zaharowic:z

Acknowledgements

It is a pleasure to aeknowledge all those who helped in this
venture: Mr. Nixon Smiley, Director, and Mr. S. Kiem, Superin-
tendent, of the Fairchild Tropical Gardens, for their .special care
in providing facilities conducive to di.scussion; Miss H. Schapiro,
Mr. B. Fritzie, Mr. H. Fernandez, Mr. D. Stafford, and Mr. E.
Zaharowicz, graduate students of the University of Miami, for their
help throughout the symposium; Mr. R. Conklin of the Miami Sea-
quarium, for being a generous host to the participants and their
families; Mrs. N. Jaffe, Mrs. E. Hirshhorn, Mr. J. Bovaird, and Mr.
H. Reasor, who, in addition to their regular responsibilities, con-
tributed in the preparation of the recorded discus.sions and of the
index of this volume; Dr. B. Strehler, for his most helpful sugges-
tions in expediting publication; Drs. E. Muscatine and E. L. Cham-
bers, for their many suggestions; and Dr. R. }. Boucek of the Howard
Hughes Medical Institute, for his encouragement and for providing
facilities for arranging the symposium. Finally, we wish to express
our deep thanks to every participant of the symposium for his
cheerful cooperation in responding to our seemingly endless requests
for corrected manuscripts, di.scussions, and galleys.

Contents

PAGE

The Fine Structltre of Cells in Hydra ... 1

Arthur Hess

The Fine Structure of Intercellular and Mesogleal
Attachments of Epithelial Cells in Hydra , . 51

Richard L. Wood

Is there a Nervous System in Hydra? .... 69

Floor Discussion

Nematocyst Development ...... 77

David B. Slautterback

The Fine Structure of the Stenoteles of Hydra . 131

George B. Chapman

Chemistry of Nematocyst Capsule and Toxin of Hydra
littoralis .......... 153

Edward S. Kline

Physalia Nematocysts and Their Toxin . . . 169

Charles E. Lane

Compounds of Pharmacological Interest in Coelen-
terates .......... 179

John H. Welsh

Page
Present State of Nematocyst Research: Types, Struc-
ture, AND Function ........ 187

Cadet Hand

Activation of the Feeding Reflex in Hydra Uftoralis . 203

Howard M. Lenhoff

The Nutrition of Hydra ...... 233

David L. Claybrook

Isolation and Maintenance in Tissue Culture of Coe-

LENTERATE CeLL LiNES ....... 245

John H. Phillips

Symbiosis in Marine and Fresh Water Coelenterates . 255

Leonard Muscatine

On the Relation of Calcification to Primary Produc-
tivity IN Reef Building Organisms ..... 269

T. F. Goreau

The Development of Cordylophora .... 287

Chandler Fulton

Developmental Problems in Campanidoria . . . 297

Sears Crowell

Patterns of Budding in the Freshwater Hydroid Cras-
pcdacusta .......... 317

Charles F. Lytle

Page
Feedback Factors Affecting Sexual Differentiation in

Hydra littoralis ......... 337

W. F. LooMis

Apparent Rhythmicity in Sexual Differentiation of

Hydra littoralis 363

Helen D. Park

Aging in Coelenterates ....... 373

Bernard L. Strehler

Studies on Chemical Inhibition of Regeneration in

Hydra 399

Robert E. Eakin

A Study of Normal and Abnormal Regeneration of

Hydra 413

Dorothy B. Spangenberg

Growth Factors in the Tissues of Hydra . . . 425

Allison L. Burnett

Nucleic Acid and Protein Changes in Budding Hydra

littoralis 441

Yu-Ying Fu Li and Howard M. Lenhoff

Index 449

The Fine Structure
of Cells in Hydra

Arthur Hess^

Department of Anatomy, Washington Univers^ity School of Medicine, St. Louis,

Missouri.

Hydra can be considered to have the following anatomical
regions: tentacles, hypostome or mouth region, column or stomach,
peduncle and basal disk. Sections of the colvmm will serve most
frequently to introduce the general histology of hydra. Then
variations of the different body regions will be presented.

Hydra has in general two cellular layers, ectoderm or epidermis
and endoderm or gastrodermis separated by a layer called mesoglea.
The ectoderm is composed basically of epithelio-muscular cells and
contains dispersed cnidoblasts or nematocyst-bearing cells and
interstitial cells or undifferentiated cells. Gland cells occur in spe-
cialized regions. The endoderm contains gland cells, digestive cells
and interstitial cells in its generalized areas. Cnidoblasts occur only
rarely in the endoderm. The mesoglea is acellular.

Whole Hydra oligactis were fixed in an extended state in
Dalton's fluid, a solution containing 1% osmium tetroxide, 1%
potassium dichromate at a pH of 7.2 to 7.6 and 0.85% sodium chlo-
ride, in an ice bath for 15-45 minutes. Sometimes, the Hydra was
divided into its various body regions; at other times, the animals
were treated as a whole. They were then dehydrated in alcohol and
embedded in methacrylate or araldite. Some sections were stained
with lead acetate or potassium permanganate. They were photo-
graphed in the electron microscope.

iThe author wishes to acknowledge the participation of Dr. A. I. Cohen and
Mrs. Dorothy Sanderson in tliis study. The author's present address is Department
of Physiology, University of Utah College of Medicine, Salt Lake City, Utah.

THE BIOLOGY OF HYDRA : 1961

GENERAL HISTOLOGY

THE ECTODERM

Epithelio-muscular cells (Figs. 1, 3). Vacuolated cells are seen
in the ectoderm. Their nuclei are large, of even granular texture,
and contain prominent nucleoli. These cells have a few double mem-
branes and many mitochondria in their cytoplasm. They frequently
contain dense inclusions, which we have not as yet identified. Small
vacuoles, in addition to the large ones, are present. Within these
cells and accumulated at their base, closely packed bundles of fine
fibrils arranged in parallel and running longitudinally with respect
to the column axis are seen. The muscle system will be considered
separately later.

These cells, frequently but not always, are the surface cells of
Hydra. The surface of Hydra is covered by a granular material
resting on two membranes (Fig. 6). One membrane obviously
belongs to the surface cells, usually epithelio-muscular, but can be,
at times, the cnidoblast. The other membrane apparently does not
belong to a cell. A short \arying distance separates the outer mem-
brane of the surface cell and the membrane on which the granular
material rests. Hydra, therefore, appears to be covered over most
of its surface by this thin cuticular material.

Interstitial cells (Figs. 4, 5). Groups of small, rounded cells
occur in the ectoderm. They are numerous in some areas and absent
from others. These appear to be interstitial cells. They are charac-
terized by having a very finely granular particulate cytoplasm with
no double membranes. Mitochondria and a Golgi apparatus are
present. Their nuclei are evenh^ granular with one or more promi-
nent nucleoli. The cells are frequently very intimately related to
each other and at times, the limiting membranes between two adja-
cent cells appear to be lacking and the cells appear to be syncytial
(Fig. 5). Since these cells give rise to cnidoblasts, some interstitial
cells can be seen with a few double membranes in their cytoplasm
suggesting that they are beginning their differentiation. These cells
can be seen at times adjacent to the muscle layer on the mesoglea.

Cnidobkists (Figs. 3, 7-10). Cnidoblasts frequently occur in
groups and can be found near the mesoglea or sometimes forming

ARTHUR HESS 3

the surface cell of Hydra. These cells have mitochondria and a
Golgi apparatus. However, it is the presence of the double mem-
branes or endoplasmic reticulum which renders these cells distinc-
tive. The cnidoblasts apparently are deri\ed from interstitial cells.
The cnidoblasts bearing \ery immature nematocysts have a series
of vesicles (Fig. 7). As the nematocyst matures, these vesicles in-
crease in amount and extent and apparently coalesce until the sys-
tem of double membranes within the cell becomes quite elaborate
and striking ( Fig. 8 ) . The nematocyst increases in size and dis-
places the nucleus. In cnidoblasts with well-developed nemato-
cysts, the double membranes begin to decrease in amount ( Fig. 9 ) .
In cnidocytes having what appear to be mature nematocysts, the
double membrane system appears to have regressed and the cyto-
plasm of the cells is again granular with only a few strands of double
membrane remaining ( Fig. 10 ) .

These cells are also apparently in syncytial relation to each other
and frequently, the cell membranes between adjacent cnidoblasts
can be seen to be lacking. Apparently the syncytium is no longer
present after the nematocysts are mature and the cnidoblasts have
completed their differentiation and are called cnidocytes. Each
mature cnidocyte appears surrounded by a complete cell membrane
in the tentacle, as will be shown later.

THE MESOGLEA

(Figs. 1, 15, 16, 19-21). The mesoglea presents a vary-
ing appearance in electron micrographs. It may appear fibrous or
granular. Some of this \ariability may be due to the state of con-
traction or extension of the Hydra during fixation. No cells are
present. Pieces of cytoplasm seen in the mesoglea can be seen to
be connected to ectoderm or endoderm cells which are pushing
into the mesoglea. These pieces of cytoplasm are surrounded by
a cell membrane and thus strictly are outside the mesoglea.

The mesoglea is apparently not surrounded by its own limiting
membrane. It penetrates between the cells of the ectoderm and
endoderm (see especially Figs. 15 and 19), and granules, similar
to those seen in the mesoglea, can be found in extracellular spaces
between ectodennal and endodermal cells (Figs. 15, 19, 23, 25).

4 THE BIOLOGY OF HYDRA : 1961

Thus, the constituent cells of Hydra can be considered as embedded
in mesoglea and the mesoglea forms a supporting substance for
the cells.

THE ENDODERM

Gland cells (Figs. 11, 12). The gland cell pours its secretion
into the lumen to break down the food and make its products avail-
able for digestion. Essentially only one kind of gland cell has been
found. This cell contains a series of large interlacing vacuoles,
which most frequently appear light, but sometimes dark. Toward
the base of the cell, the vacuoles frequently are smaller than in
the portion of cell near the lumen. The cell appears to be under-
going a process of manufacture of the vacuoles starting toward the
base. Thus, various vacuolar arrangements can be seen, but they
are believed to be stages in the appearance of a single kind of gland
cell. Between the vacuoles, some mitochondria and double mem-
branes appear. Toward the base of the cell, the vacuoles are not
present and the cytoplasm is filled with mitochondria and double
membranes. It is probably here where the manufacture of the
vacuolar contents, which will be secreted into the lumen, begins.
The nucleus of the cell is toward its base. This cell apparently does
not rest upon the mesoglea.

The digestive cell (Figs. 13-16). The digestive cell absorbs
the food products after action of the gland cell. The digestive cell
also undergoes cyclical changes according to the feeding activities
of Hydra and also contains various inclusions depending on the kind
of food and time of feeding. The cell can appear columnar and
rather well organized or can contain huge vacuoles. It has a light
cytoplasm with mitochondria and a Golgi apparatus. The surface
of the digestive cell usually has a series of small cytoplasmic projec-
tions or villi extending into the lumen (Fig. 14). The digestiv^e
cells contain the endodermal muscle filaments at their base and
rest upon the mesoglea (Figs. 15, 16).

Flagella (Fig. 18). Apparently both gland cell and digestive
cell have flagella. It is difficult to determine exactly how many
project from each cell. Two to four flagella are commonly seen.
The flagella present the nine peripheral and two central longitudinal

ARTHUR HESS 5

filaments characteristic of motile flagella in other animals. These
flagella differ slightly from those of other organisms in that they
possess a relatively thick membrane surrounding the filaments which
frequently becomes separated from the filaments so that its rela-
tion to the filaments does not appear as intimate as the relatively
thin membrane enclosing flagella elsewhere.

THE MUSCULAR SYSTEM

(Figs. 1, 15, 16, 17, 20, 21). The ectodermal muscle layer runs
essentially longitudinally, while the endodermal layer is predom-
inantly transversely oriented. The muscle filaments contained
as a cell organelle in the base of the epithelio-muscular and diges-
tive cells run parallel to each other, appear to be essentially of
one kind, present no cross striation, and hence can be considered
as smooth muscle filaments. The muscle fibers run along the meso-
glea. They appear to be anchored to the mesoglea by small cyto-
plasmic extensions of the cells containing them (see especially Figs.
15 and 16 ) . These extensions are frequently more numerous and
robust on the ectodermal side and sometimes muscle filaments
extend into these cytoplasmic attachment roots. The ectodermal
muscle filaments in the cytoplasmic extensions of the base of one
epithelio-muscular cell are very intimately related to the muscle
filaments of an adjacent epithelio-muscular cell. The extensions of
the cells can dovetail with each other in finger-like extensions or
can overlap each other. However, the filaments do not pass from
one cell to another. The filaments sometimes appear to insert on
the cell membrane and when this happens in adjacent cells, an
apparent thickening of the adjacent cell membranes occurs and a
desmosome-like effect is produced ( Fig. 17 ) . The digestive cells
usually do not undergo such an intimate arrangement and adjacent
digestive cells are related to each other by relatively smooth
membranes.

There are points along which the mesoglea appears very thin
or interrupted and where the ectoderm and endodermal muscle
filaments, or at least the membranes of the cells containing them,
are practically in contiguity (Figs. 20, 21). Probably some very
thin mesogleal substance intervenes between them since, as men-

6 THE BIOLOGY OF HYDRA : 1961

tioned above, all the eells are probably embedded in mesoglea.
These points of contact between the muscle layers are fairly fre-
quent and occur in all areas investigated.

THE RELATIONS OF CELLS TO EACH OTHER

The special relationships of muscle cells and the fact that all
cells appear embedded in mesoglea have already been discussed.
However, there are other peculiar relations of cells that should be
mentioned. By no means are the limiting membranes of the cells
smooth. At times, a button or snap fastener arrangement can be
seen where one cell evaginates a piece of cytoplasm to rest in an
indentation of an adjacent cell ( Fig. 2 ) . This causes the frequent
appearance of circular areas of cytoplasm located between cells.
In addition, terminal bars are seen between some cells lining the
lumen (Fig. 14) and between other cells near the mesoglea
(Fig. 19).

NERVE CELLS AND FIBERS

No cell was foimd which could be called a nerve cell. As
explained above, the small circles located between cells, which
may sometimes form clusters and appear like bundles of nerve
fibers (Figs. 15, 18, 21, 26), probably result from the peculiar
formations of the cell borders. There is the possibility that
nerve tissue of Hydra may appear different in the electron micro-
scope from that of other organisms, and we are thus unable to
identify nerve cells or fibers in our electron micrographs. However,
if the absence of nerve tissue in Hydra can be accepted, one may
perhaps go further and wonder if, indeed. Hydra needs any nerves.
The epithelio-muscular cells containing the muscle filaments are
on the surface of the animal and there can act as receptor cells
to lead the impulse to its muscle filament organelles. The impulse of
one ectodermal muscle fiber could easily be transmitted to muscle
filaments in adjacent epithelio-muscular cells. Endodermal muscle
could conduct an impulse from one cell to another in a similar
manner. Lastly, the interaction of ectodermal and endodermal
muscle could well be achieved through the points where mesoglea

AHTUL'R HESS

is practically absent and the two muscle layers are essentially in
contact. The ordinary slow moxement which Hydra performs could
well be subserved bv muscle to muscle transmission.

REGIONAL HISTOLOGY

llic Jiypvstoinc (Figs. 26, 27). The hypostome has a relatively
low-lying ectoderm (Fig. 26). The endoderm is extremely well-
developed ( Fig. 27 ) . The very large cells and dense accumulations
of gland cells sometimes practically obliterate the lumen.

The peduncle. The endoderm of the peduncle is reduced in
extent and gland cells are absent. The digestixe cells consist of
very vacuolated thin strands of cytoplasm. The ectoderm is similar
to that already described. The epithelio-muscular cells of this region
are characterized b\- ha\"ing granules near their surface (see
Fig. 30).

T]u' based disk (Fig. 29). The endoderm of this area is like
that of the peduncle. The ectoderm is characterized b\- the presence
of a type of gland cell which consists mostl)- of double membranes
and has large granules, similar to those seen in the epithelio-mus-
cular cells of the peduncle, l)ut much larger. These granules of the
gland cells of the ectoderm in this area are apparently the substance
produced to cement Hydra to the substratum. The ectodermal
cells of the pedal disk have small extensions of cytoplasm or villi
on their surface. No granular and cuticular material is present on
the surface of Hydra at this level.

Hie tentacle (Figs. 22, 24, 28). The tentacle arises at the level
of the Inpostome. Sections through this region reveal a gradual
change of the cells with the tentacle compared to the hypostome
having a reduced endoderm and ectoderm, reduced number of
gland cells, increased vacuolation of digestive cells, increased num-
ber of cnidocytes, and perhaps better de\ elopment of the muscle
filaments in the epithelio-muscular cells. The endoderm-mesoglea
interface at this level exhibits a characteristic scalloped appearance
(Fig. 28).

The endoderm of the tentacle is severely reduced (Fig. 22). It
consists of \'ery thin wisps of cytoplasm of digestive cells enclos-

8 THE BIOLOGY OF HYDRA : 1961

ing huge vacuoles. The ectoderm is also very thin and has a series
of bulges or ridges. At the height of each ridge are present the
cnidocytes containing apparently mature nematocysts (Fig. 22).
The cnidocytes can be surrounded by the cytoplasm of epithelio-
muscular cells. Sometimes the cnidocytes rest on the muscle layer,
in which case, epithelio-muscular cell cytoplasm is on three sides
of them (Figs. 22, 24). At other times, the cnidocyte is the surface
cell (Fig. 24). However, as far as we can determine, each cni-
docyte is enclosed by a complete cell membrane, even when one
cnidocyte abuts against another (Fig. 24). Hence, the syncytial
relationship of cnidoblasts with immature nematocysts seen in the
column has broken down during the maturation of these cells seen
as cnidocytes in the tentacle. Frequently, muscle filaments are
present in the cnidocyte (Fig. 24). At what stage of cnidoblast
development these muscle filaments make their appearance is
unknown.

The bud (Fig. 30). We have not studied the bud in detail, but
we have noticed that the ectodermal cells of mother and bud fuse
insensibly with the cuticular layer of the mother continuous over
the surface of the bud. The portion of bud attaching to the mother
Hydra looks essentially like the peduncle of the mother Hydra with
very vacuolated endodermal digestive cells and ectodermal epithe-
lio-muscular cells containing granules near their surface.

Pertinent literature is cited in:
Hess, A., A. I. Cohen, and E. A. Robson. 1957. Observations on the structure

of hydra as seen with the electron and hght microscopes. Quart. }. Microscop.

Sci. 98: 315-326.

EXPLANATION OF PLATES
All photographs are electron micrographs of Hydra. The line on the
photographs indicates 1/j,.

PLATE I. Fig. 1. Cross section of the ectoderm showing the epithelio-
muscular cells with their nuclei (N), inclusion bodies (I) frequently found in
these cells, the muscle filaments at the base of the epithelio-muscular cell
forming the ectodermal muscular layer (E), and the vacuoles (V) in the cells.
M is the mesoglea.

Fig. 2. The arrows point to the "snap-fastener" relationship between two
cells in the ectoderm where a portion of cytoplasm of one cell evaginates and
indents an adjacent cell.

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1 4

lit wl/^\"^^?^ ";\ '^^

fcr-^

.//;■

>t v.-

PLATE II. Fig. 3. Cross section of the ectoderm (surface of animal to
to the left) showing epithelio-muscular cells (E), interstitial cells (I) and
cnidoblasts (C), the latter in apparent syncytial relationship and having
nematocysts and prominent double membranes.

PLATE III. Fig. 4. An interstitial cell with its nucleus, granular cyto-
plasm, small mitochondria and a Golgi apparatus (G).

Fig. 5. The line, indicating the magnification, passes through an appar-
ent cytoplasmic bridge between two adjacent interstitial cells.

Fig. 6. The surface of Hydra showing the cuticular substance resting
on a membrane (Arrow # 1). Arrow # 2 shows another membrane, probably
the limiting membrane of the surface cells.

-^iim^m.

PLATE IV. Fig. 7. Cnidoblasts in syncytial relationship during the be-
ginning of nematocyst development. Double membranes are present in the
cytoplasm. See figures 8 to 10.

Fig. 8. Cnidoblasts in syncytial relationship at the height of nematocyst
development. The double membranes in the cytoplasm have increased in
amount and are very conspicuous in the cell.

•*-*

PLATE V. Fig. 9. Cnidoblast with a well-developed nematocyst. The
double membranes are present, but are regressing in amount. See figure 8.

Fig. 10. Cnidocyte (mature cnidoblast) which contains a fully-developed
nematocyst (not seen in figure). The double membranes in the cytoplasm
are severely reduced in amount and only a few strands are left. G is a Golgi
apparatus.

•"' •» ,

" 1'

-:Sf'-''

"G.

10 "

PLATE VI. Fig. 11. A gland cell with light vacuoles and concentrations
of double membranes toward the base of the cell around its nucleus. The
lumen is to the right.

Fig. 12. Dark vacuolar contents in another gland cell. Gland cells
with dark vacuolar contents are seen only rarely.

'^ }}.ri'I^

PLATE VII. Fig. 13. Digestive cells with relatively small vacuoles and
large dark inclusion bodies, probably lipid. The nuclei of digestive cells and
fairly light cytoplasm with mitochondria and some double membranes can
also be seen.

Fig. 14. The surfaces of two digestive cells lining the lumen. The cells
contain inclusion bodies. The cell membranes at the junction of the two
cells near the lumen are rather dense for a short distance and resemble a
terminal bar. Short tortuous process of cytoplasm extend into the lumen.

Plate VIII. Fig. 15. Sections showing the relations of ectoderm (EC),
endoderm (EN) and mesoglea (M). The endoderm cells have muscle filaments
in their base and send processes into the mesoglea. If the origin of the
processes is missed in section, the processes appear to be lying as organelles
or inclusions in the mesoglea. The mesoglea has no membrane around it. If
the cell membranes of adjacent cells are traced for a short distance from the
mesoglea, collections of granules, similar to those in the mesoglea, can be
found in the extracellular space (arrow).

J ^

i, I

i /

JOI'

f' W ^ / i V /.

.#*•"

PLATE IX. Fig. 16. The ectoderm is at the top, the endoderm at the
bottom and separated from each other by mesoglea. A rather robust process
passes from an endodermal cell into the mesoglea.

Fig. 17. Longitudinal section of the junction of two muscle fibers in the
ectoderm. The alternating light and dark densities on adjacent cell mem-
branes yield a desmosome-like effect. Muscle filaments do not pass from
one cell to the other.

Fig. 18. Flagella in the lumen of the hypostome surrounded by a mem-
brane and exhibiting the characteristic pattern of filaments.

T":

-. .- /

'■•■ J

■^q'

^ ■

■»_^,.

fcrf-^assfcsof

'^» ^>Cjr'" ■

,^.h.a

' 0-

^^•'V-

,Wi_

»£

<v,,.

.-^■

PLATE X. Fig. 19. The mesoglea (M) has granules and some very thin
filaments. On the ectodermal (EC) side, it can be clearly seen that the
mesoglea is not surrounded by a membrane, but rather extends between the
membranes of adjacent ectodermal cells (arrows on the ectodermal side).
The membranes of adjacent endodermal (EN) cells can be followed for a
considerable distance. The membranes are very dense as they proceed from
the mesoglea yielding a terminal bar effect. As the membranes are fol-
lowed, accumulations of granules, similar to those seen in the mesoglea, can
be found in the extracellular spaces (arrows on the endodermal side).

PLATE Xi. Figs. 20 and 21. Points of contact between ectodermal (EC)
and endodermal (EN) muscle where the mesoglea (M) is severely deficient or
essentially lacking. Figure 21 is in the region of the hypostome.

^ EC

4>i

PLATE XII. Fig. 22. A ridge or low elevation in the ectoderm of the
tentacle containing cnidocytes (C) apparently embedded in the cytoplasm
of an epithelio-muscular cell (EP). A thin mesogleal layer (arrow) separates
the ectodermal cells from the attenuated, highly vacuolated endoderm (EN).

Fig. 23. An extracellular space containing granules and enclosed by the
cell membranes of four endoderm cells, yielding a star-shaped effect.

ti EP'

•■\ ■♦. ; ..

i«

PLATE XIII. Fig. 24. Cnidocytes in the tentacle embedded in the cyto-
plasm of epithelio-muscular cells (EP). The cnidocytes are not syncytial, but
are enclosed individually in their limiting membranes. One of the cnidocytes
has muscle filaments (M). S is the surface of the animal. Near S, a cnidocil
is seen.

Fig. 25. An extracellular space containing granules and enclosed by the
membranes of endoderm cells and yielding a star-shaped effect.

5*iiS?^«*^1

-M

•#»%.

.-' A

..■^

PLATE XIV. Fig. 26. Section through the hypostome showing relations of
ectoderm, endoderm and mesoglea (M). The epithelio-muscular cell (EP)
contains vacuoles, organelles and inclusions as described previously and
muscle filaments in its base. The digestive cells of the endoderm (EN) con-
tain vacuoles, organelles and inclusion bodies and have muscle filaments in
their base. S is the surface of the animal.

.■..•'A'Sf,

«^'

^•

». ■*

:r^^:^-^^

Iv.^x ;^«^

f -•

PLATE XV. Fig. 27. Section through the endoderm of the hypostome.
Gland cells (G), with their vacuoles and digestive cells (D) containing inclu-
sion bodies, probably absorbed food, line the lumen (L). The flagella and
cytoplasmic processes extending from these cells are seen in the lumen.

PLATE XVI. Fig. 28. Section through the junction of the hypostome and
the tentacle. The arrows indicate the approximate plane of attachment of
the tentacle (on the right of the arrows) to the hypostome. The interface of
the endodermal cells (EN) and the mesoglea (M) has a characteristic scal-
loped appearance. Muscle filaments on the epithlio-muscular cells are per-
haps better developd in the tentacle than in the hypostome.

^ I h

'/A

%>^:

i '"' k-

^ /

i / I

' / /

/ /

-■^a*C

; 3Q§

IIhI

•

'< J

PLATE XVII. Fig. 29. The gland cells of the ectoderm of the pedal disk.
There is no cuticular substance on the surface (S). Small cytoplasmic proc-
esses extend from the surface of the cells. The granules (G) are probably the
secretion manufactured by these cells, especially those with more double
membranes and fev/er and smaller granules farther from the surface, to
cement Hydra to the substratum.

I i

:*«?

^-V

\..^.

J ^

•#v

•:>>'

n .>%^ ",

PLATE XVIII. Fig. 30. Place of origin of bud from mother. The arrow
indicates approximately the point of attachment of the bud to the mother.
Epithelio-muscular cells of the mother are on the bottom of the photograph.
The more highly vacuolated epithelio-muscular cells of the bud are similar
to the epithelio-muscular cells seen at the level of the peduncle. Similarly,
the granules seen near the surface of the epithelio-muscular cells of both
mother and bud are characteristic of epithelio-muscular cells of the
peduncle. The cuticular layer is continuous over the surfaces of bud and
mother.

'S'«?.^'*:%3**^ ^r

"t-^SN

~V^->^

\ (

^--v,

ic •i-'

■ *!

!%i.tr.5^.: -

-■^

^3..^.:

44 THE BIOLOGY OF HYDRA : 1961

DISCUSSION

LOOMIS: At what point do interstitial cells start differentiating
the four types of nematocysts?

HESS: I'm sure someone else later in the program could answer
that. I have not actually worked on the structure of the nematocyst
per se, just the cnidoblast.

SLAUTTERBACK: Dr. Hess, I'm afraid your fine micrographs have
stolen the thunder from the rest of the electron microscopists here.
I did not want to raise the issue of what you have called the
gland cell. I believe that on the basis of location, staining proper-
ties and appearance of the secretory granules in electron micro-
graphs, your term includes two distinct cell types as has been sug-
gested in the classical literature. We have been calling the cell
which is more prominent in the hypostome region and resembles
the goblet cell of the vertebrate digestive system, a mucous cell.
The other type, which is more prominent below the hypos'come and
resembles the pancreatic acinar cell, we have called the zymogenic
cell.

HESS: It seemed to me that these different appearances might be
cyclical changes. Most of the cells have light vacuoles and only
rarely do some of them stain darker in the electron microscope.
I haven't done histochemical staining, and you might be right that
two different cell types occur because many people speak of these
two kinds of cells.

BURNETT: I would like to mention some histochemical results
we have obtained on regenerating hydra. If the hypostome of the
hydra is excised, we find that mucous cells begin to appear in
abundance in the gastrodermis at the point of excision about 12-18
hours after cutting. The secretory material in these cells is PAS
positive, stains with alcian blue, is metachromatic after toluidine
blue or methylene blue staining, and is removable by hyaluronidase
digestion. This material is most certainly an acid mucopolysac-
charide. Gland cells appear six hours after excision. The secretory
droplets in these cells are several times larger than those found
in the mucous cells. Moreover, gland cells do not stain with alcian

ARTHUR HESS 45

blue and are not metachromatic, but positive to Millon's reaction
for proteins. These two types of cells are, therefore, quite different
from one another both histochemically and morphologically.

I have a question. Were the cnidoblasts in the same cluster
forming the same type of nematocysts?

HESS: I didn't notice the type of nematocyst, but all those within
a cluster seem to be in the same stage of development.

FAWCETT: I would like to comment on that point. It has been
our experience that within any single cluster of cnidoblasts, they
are all forming nematocysts of the same kind. They are also pre-
cisely synchronized in their development. I would comment further,
if I may, on the syncytial relationship that was mentioned. I noticed
in Dr. Hess' pictures two distinct kinds of syncytial relationships.
In a number of instances, the connections between cells appeared
simply as small discontinuities of varying lengths in the pairs of
membranes constituting the boundaries between cells. We have
seen such apparent communications, but although our technique
was seemingly good enough to make it unlikely that these were
artifactitious breaks in the continuity of the cell membranes, this
has nevertheless always been a disturbing possibility. There is an-
other kind of syncytial relationship between cnidoblasts which is
clearly not artifactual, and is of considerable interest in relation
to the mechanism of cell division and the control of differentiation.
It is this kind of intercellular bridge, found in both interstitial
cells ( Fig. 1 ) and cnidoblasts ( Fig. 2 ) , that I would like to illustrate
in order to emphasize the special nature and probable significance
of the syncytial relationship between cnidoblasts. Groups of eight
or sixteen cells arising by proliferation from a single interstitial
cell remain connected by bridges a micron or so in diameter, en-
closed by a specialized, thickened area of membrane that has a
characteristic contour. There is no possibility that this localized
thickening of the plasmalemma and special configuration of the
sin-face could arise as an artifact of specimen preparation. Notice
the heartshaped outline of the intercellular space and the definite
ridge that encircles the waist of the intercellular connection. Dr.
Slautterback and I believe that such bridges arise during division
of the interstitial cells when the constricting cleavage furrow en-

.-/ji

Fig. 1. Intercellular bridge of interstitial cells.

counters the spindle remnant, and is arrested by it for a time. This
occurs very commonly in mitotic divisions in many kinds of genninal
and somatic cells and gives rise to a transient structure called a
spindle bridge. Usually, however, such connections between the
daughter cells endure only for several minutes and then when the
spindle remnants have resorbed the cleavage is completed. Evi-
dently in the case being described here, cleavage does not resume
and absorption of the spindle filaments leaves the daughter cells in
open communication through short cylindrical bridges large enough
to permit mitochondria and other formed elements of the cytoplasm
to pass from one cell body to another. As a consequence of the
matter in which they are formed, there is never more than one

Fig. 2. Intercellular bridge of cnidoblasts.

I Nematoc^t

p^.

nterceltular
Bridge

Nematocyst

48 THE BIOLOGY OF HYDRA : 1961

such bridge between any two cells in the cnidoblast cluster. The
bridges persist throughout the period of differentiation of the nema-
tocysts. If the nematocysts are eventually to migrate as individual
cells, the bridges connecting them must be severed at some time
late in their differentiation, but this process has not yet been ob-
served. We believe that the syncytial relationship of the cnidoblasts
is probably the morphological basis for the synchrony of their differ-
entiation. It is interesting that the same kind of synchrony is seen
in the groups of developing germ cells in the testes and these are
also connected by intercellular bridges that form in the same way.

CLAYBROOK: Do either of you find cytoplasmic bridges between
different cell types, or are they only between two of the same kind?

HESS: I've only seen them between the same cell type. How about
you?

FAWCETT: Bridges of the kind I have been describing occur only
between cells of the same type.

HESS: I've seen a break in the cell membranes of the spermatids,
like the first type of interconnection of which you spoke. We
thought that it was an artifact until we saw cytoplasm and mito-
chondria in the intercellular bridge running between the two syncy-
tial cells.

GAUTHIER: May we return to the subject of gland cells? If the
two cell types represent only a cyclical change in one cell type,
would you expect that starvation might produce a levelling off so
that only one type would be present?

HESS: Well, I thought that the different appearances of gland cells
indicated cyclical changes of one cell type, but others here appar-
ently disagree.

GAUTHIER: In preliminary experiments with starved hydra, I
have found that two distinct types of gland cells persist for as long
as twelve days,

GOREAU: I am interested in the so-called microvilli you have
shown. We have seen microvilli in gorgonian and scleractinian
material which have a much more regular and permanent appear-

ARTHUR HESS 49

ance than anything you ha\'e shown. The processes in your sections
of Hydra epidermis look to me hke temporary cytoplasmic pseudo-
podia. They certainly don't have the same well organized distribu-
tion that is seen, for example, in the epidermal cells of corals where
the microvilli are arranged in a regular ring around the base of the
flagella (Goreau and Philpott, 1956. Exptl. Cell Research 10:552).
I'm also interested to see that the epidermal cells of Hydra are not
flagellated.

HESS: All the flagella of Hydra arise from endodermal cells and
extend into the lumen.

GOREAU: We've never found more than one flagellum per cell,
whereas you seem to think there are more than one.

HESS: Yes, in the gastrodermis, each cell apparently has from two
to four flagella.

The Fine Structure of Intercellular
and Mesogleal Attachments
of Epithelial Cells in Hydra

Richard L. Wood

Department of Anatotny, University of WasJiington Scliool of Mediciiie, Seattle.

Cellular interactions in multicellular organisms have been ex-
amined both from the physiological and the morphological
points of view. As a result of these studies it has become clear that
there are certain general features of epithelia which are related to
special kinds of adhesive properties. It is further realized that
these special adhesive properties are not distributed uniformly over
the cell surface. The epithelial layers of hydra share these general
properties of epithelia, although the details of intercellular attach-
ment sites seem not to have been studied extensively in the past.

Hydra consists essentially of a bicellular leaflet of epithelia and,
therefore, is well suited for studies of epithelial cell interactions. The
epithelia of hydra are perfectly good epithelia, but at the same
time the individual cells serve several functions, many of which are
not usually associated with functions of epithelium in a single layer
in higher organisms. The presence of well developed terminal bar
type attachment areas between these epithelial cells of hydra is
certainly to be expected from our knowledge of higher organisms.
Such areas do occur and the detailed structure differs from previ-
ously described intercellular attachments.

Basal processes of many epithelial cells in hydra contain muscle
fibers. Special relationships between adjacent muscle fibers and

^This research was aided in part by Grant No. H-2698 from the National Institutes
of Health, Public Health Service.

52 THE BIOLOGY OF HYDRA : 1961

between muscle elements and connective tissue, or mesoglea, would
also be expected, and indeed they also occur. The purpose of this
presentation is to review some of these relationships as I have
observed them using light and electron microscopy. These observa-
tions pose a great number of additional questions which will require
some new approaches for further elucidation.

In this presentation I will refer to the intercellular attachments
as desmosomes. I prefer desmosome as a general descriptive term
for intercellular attachments because the term was originally pro-
posed with a recognition of the functional relationship and basic
similarity of the various forms of intercellular attachment (9).
The concept of desmosome (literally "bonding body") seems to be
well substantiated by micromanipulation experiments with various
kinds of epithelium from different organisms.

The present observations were made on specimens of Chloro-
hydra viridissima and Pelmafohijdra oligactis. Material was fixed
in osmium tetroxide buffered in acetate-veronal (6) or s-collidine
(1) at pH 7.4. The tissue was dehydrated in ethyl alcohol and
embedded in a mixture of n-butyl and methyl methacrylates or in
either Araldite or Epon epoxy resin (see Luft, ref. 5). Light
micrographs were made from one micron sections cut from epoxy
embedded blocks and stained according to the method of Rich-
ardson, et al. (7). The electron microscopy was done on an RCA-
2C with an improved power supply and with a Siemens Elmiskop I.

The epithelial layers of hydra mostly consist of single layers
of cuboidal to columnar epithelial cells. In the epidermis interstitial
cells occur between the epithelial cells near their bases and nema-
tocytes occur between epithelial cells at the outer surface of the
animal. The gastroderm contains two easily identifiable cell types,
nutrient cells and glandular cells. A thin lamella of mesoglea sepa-
rates the two epithelial layers. This general configuration is dem-
onstrated in the first illustration. Figure la is a light micrograph of
a transverse section through the region of the hypostome in
Chlorohydra. Glandular cells and basally located intracellular
symbiotic Zoochlorellu may be identified in the gastrodermis.
Light areas near the mesoglea at the base of the epidermal cells
represent cross sections of muscle fibers. Figure \h shows a trans-
verse section through the column of Pelmatohydra. The larger

"•P* ^Sk 7^* "* " *

r -/"Ifc « ^ _.

Fig. 1. Light microscope pictures of a, Chlorohydra and b, Pelmatohydra.
In a the epidermis is at the top and the gastrodermis at the bottom. The two
layers are separated by the mesolamella along which muscle fibers may be
seen. In b the epidermis is at the right and the gastrodermis at the left with
the mesolamella between. Note the obvious muscle fibers at the base of the
epidermis and the connection between epithelia at the circle. Desmosomes
appear at the arrows. The black circular objects in the gastrodermis of a
are Zoochlorella; in b similar structures are food particles. C, cnidoblasts;
N, nucleus, o— 2200X. fc— 2200X.

54 THE BIOLOGY OF HYDRA : 1961

size and lack of Zoochlorella make Pelmotohi/dra easier to ex-
amine. At the free outer surface, adjacent epidermal cells are
bound together by terminal bar type desmosomes ( arrows ) . These
desmosomes were not described in earlier light microscope studies
of hydra or in more recent electron microscope studies by other
workers (2, 4, 11). Interstitial cells, nematoblasts and gastroderm-
al nutrient cells are seen clearly in Figure lb.

These general features of hydra epithelia are shown to even
better advantage in low magnification electron micrographs. Figure
2 is an electron micrograph of a section through the gastric region
of Chlorohijdra. The prominent dense bodies in the gastro-
dermal cells are Zoochlorella. Other identifiable features include
nuclei, microvilli, other cellular inclusions and muscle processes.
The two epithelial layers are separated by the thin mesolamella
which appears dense in this picture. Desmosomes appear as areas
of increased density between adjacent cell surfaces, especially near
the outer surface of epidermal cells and the lumenal surface of
gastrodermal cells (arrows). Similar densities occur between
adjacent membranes of interdigitated muscle processes (Fig. 2,
circle ) .

In both species of hydra examined the desmosomes which are
present near the free surfaces of epidermal and gastrodermal cells
display a very complex morphology when viewed at higher magni-
fication. The two apposed plasma membranes each exhibit the
dual profile of the "unit membrane" of Robertson (8), the two
peaks of density being about 70 Angstrom units apart. The increase
in density noted by light microscopy and in lower magnification
electron micrographs is seen to be due to a condensation of intracel-
lular material and to the presence of a specially oriented intercel-
lular matrix. These features are shown in Figure 3, a and b, an
example of an epidermal desmosome in a specimen prepared in
the usual way and then stained with phosphotungstic acid prior to
embedding. In this preparation the junction of at least three differ-
ent epidermal cells is represented. The condensation of intracellular
material appears somewhat vague at this magnification but the
organization of intercellular material is well demonstrated. The
two apposed cell surfaces are connected directly by a series of
parallel densities oriented perpendicular to the plane of the plasma

RICHARD L. WOOD

membranes. The intercellular space is thereby divided into a series
of compartments.

From examination of oblique or longitudinal sections of these
desmosomes it is clear that the intercellular connections are not

33t^

y9i

Fig. 2. Low magnification electron micrograph of Chlorohydra. The epi-
dermis is at the top and the gastrodermis at the bottom. Zoochlorella appear
in the gastrodermal cells. Cross sections of muscle fibers lie adjacent to the
mesolamella in the epidermis. Desmosomes are apparent in both the epi-
dermis and the gastrodermis (arrows). Specialized muscle-to-muscle attach-
ment is indicated by increased densities such as at the small circle. Note the
large intracellular vacuoles in both epithelial layers. V, intracellular vacuole.
2700X. (Originally published in J. Biophysic. and Biochem. Cytol., 6:
343-352, 1959).

Plasma membrane

0.1//

Fig. 3. An epidermal septate desmosome of Pelamatohydra. The junction
of three cells in a shows the reflection of ceil surfaces into the attachment
region (arrow) and the prominent cross connections. At b the lower central
portion of a (framed) is shown at higher magnification. At the double arrow
the outer dense component of the lower plasma membrane appears to be
continuous with the dense lines of the intercellular septa. The diagram at c
illustrates the arrangement of septate desmosomes as visualized from these
observations. See text, o— 53,000X. 6— 130,000X. (These illustrations origi-
nally appeared in J. Biophysic. and Biocbem. Cytol., 6: 343-352, 1959).

Fig. 4. a. End-to-end apposition of muscle fibers in the epidermis of
Pelmatohydra. Note the irregular line of contact and the increased density
associated with the two cell surfaces (arrows). The myofilaments appear as
small streaks oriented towards the attachment zone. The subjacent mesoglea
exhibits very fine filaments more or less randomly arranged, b. End-to-end
apposition of gastrodermal muscle fibers at high magnification. The fila-
ments in gastrodermal muscle appear less conspicuous than those in the epi-
dermis. M, mitochondrion; ME, mesoglea. a — 17,000X. b — 80,000X.

58 THE BIOLOGY OF HYDRA : 1961

simple bars but actually form lamellar partitions, or septa. The exact
nature of the septa is not yet clear but there is some indication
that they may be continuous with the outer dense components of
the two apposed "unit" membranes (Fig. Sb, arrow). A diagram-
matic representation of this type of desmosome is shown in Figure
3c. The two plasma membranes are joined by septa which may
possibly have direct connections to the outer components of the
apposed plasma membranes. Lack of continuity, as illustrated at
B, is more commonly seen than continuity shown at A, so it is
uncertain which configuration is more accurate. Perhaps both
conditions occur along the course of the same septum.

Another type of intercellular attachment occurs in hydra where
muscle processes are apposed end to end. Myofilaments appear to
insert into regions of increased density and the two cell surfaces
are maintained always in close approximation. This relationship,
shown in Figure 4 (a, h) resembles the intercalated disc of verte-
brate cardiac muscle. The intercalated disc is now recognized as
a kind of desmosome (see Sjostrand and Andersson, ref. 10).
This type of attachment is particularly clear in longitudinal sections
of the epidermis (Fig. 4«). In cross section they appear at the
base of the gastrodermis and may be distinguished as irregu-
lar, dark streaks in light micrographs (Fig. \h).

In the basal region of the tentacles, and in the upper part of the
column, there is a special type of relationship of the muscle pro-
cesses to the mesoglea. This type of attachment may also be identi-
fied by light microscopy in favorable preparations. Figure 5a is a
light micrograph of a longitudinal section of a tentacle near its
junction with the hypostome. Near the mesoglea an area of in-
creased density is quite apparent, but the details of its structure
are not obvious. A similar region viewed in the electron microscope
(Fig. 5i>) shows that the density is caused by a specialized
muscle insertion on mesoglea. The attachment is accomplished by
means of a narrow finger of epitheliomuscular cell cytoplasm which
becomes intimately associated with an area of increased density in
the adjacent mesoglea. The cytoplasmic finger contains a condensa-
tion of material which appears to be organized into a series of
small tubular elements arranged at right angles to the plane of the
plasma membrane (Fig. 6). The disposition of these tubules sug-

Fig. 5. Attachment of muscle to mesoglea in Pelmatohydra; o is a light
micrograph of a longitudinal section of a tentacle. The epidermis is at the
left and its scalloped surface indicates partial contraction of the tentacle.
The dense line at the base of the epidermis (arrow) indicates a specialized
form of attachment of muscle to mesoglea. A similar region viewed in cross
section with the electron microscope is shown at b. Epidermal muscle fibers
lie adjacent to the mesoglea. An extension of one muscle fiber becomes as-
sociated with a projection of mesoglea. See text. L, lumen; N, nucleus; M,
muscle; ME, mesoglea. o— 2,000X. 6— 20,500X.

THE BIOLOGY OF HYDRA : 1981

gests a supporting function such as might be required in areas where
there is increased mechanical stress.

The final example of a possible attachment mechanism in
hydra which I will present is another arrangement of epithelial
cell surfaces at the level of the mesoglea. In my preparations, both

Fig. 6. High magnification of muscle attachment to mesoglea at a ten-
tacle base in Pelmatobydra. The mesoglea is to the right. The cytoplasmic
finger of the muscle fiber extends vertically through the center of the pic-
ture. Note the transversely oriented tubular structures (arrow) and the pat-
terns of increased density. See text. ME, mesoglea. 120,000X.

RICHARD L. WOOD 61

Fig. 7. Mesogleal relationship of epidermal muscle processes (top). The
muscle fibers extend irregular processes into the mesoglea, some of which
traverse the mesoglea completely (center). Those which traverse the mes-
oglea may abut against similar processes from the gastrodermal cells (bot-
tom). C, cnidoblast; M, muscle. 7,000X.

for light microscopy and for electron microscopy, the mesogleal
sm'face of epitheliomuscular cells is plicated and irregular. Fre-
quently the mesoglea is completely traversed by narrow cytoplas-
mic processes (Fig. lb, circle; Fig. 7). These connections were seen
and illustrated by Hadzi in 1909 (3) but have not captured the
attention of morphologists again until rather recently. They extend
from epithelial cells situated in both layers. Within the mesoglea, or
at one epithelial surface, the processes may abut against the oppo-
site epithelium either along a fairly broad surface or in a very
limited area (Figs. 7,8). So far as has been observed, the processes
extending across mesoglea represent regions of contact between
the two epithelial layers but not cytoplasmic continuity. Two dis-
tinct plasma membranes have always been seen although a reduc-
tion of the spacing between the apposed membranes is often evident.
In fact, the typical 150-200 A separation may essentially disappear,
as is illustrated in Figure 8.

62 THE BIOLOGY OF HYDRA : 1961

The irregular profile of epithelial cell surfaces being presented
to the mesoglea could possibly reflect a mode of insertion into the
extracellular matrix, as suggested by Hess, Cohen and Robson (4).
Cell contacts across the mesoglea could be related to an attachment
function but could also be related to the transfer of nutrients from
gastrodermis to epidermis or to some mechanism of direct integra-
tion between the two muscle layers.

In this paper I have attempted to present a brief account of
some of the various types of attachment that occur between epithe-
lial cells and between the epithelial cells and mesoglea in hydra.
The conclusion that all of these specializations represent kinds of
cellular attachment is based on comparison with other organisms
and on attempts to correlate structure with function. These attempts
take into consideration special physiological problems related to the
fresh water environment and the mode of feeding of these organisms.
A permeability barrier for the organism seems essential and attempts
to find a structural basis for this barrier have been unsuccessful in
the past. I have postulated that the septate form of desmosome could
be important in preventing the influx of excessive fluid to the inter-
cellular spaces (12). There is no direct evidence, however, that
septate desmosomes are any more effective in this respect than are
ordinary terminal bars found in ductile epithelium or gut of higher
forms. In fact, I am not sure that one can say positively that term-
inal bars function to preserve the intercellular milieu in any situa-
tion but evidence seems to favor such an interpretation.

At end-to-end and lateral contacts of interdigitating muscle
fibers a strong adhesion is something which would appear essential
for the efficient transmission of force during contraction of the
muscle fibers. By the same token, the special kinds of attachments
of muscle to mesoglea might be expected in areas of particular
stress, such as presumably occurs at the bases of the tentacles.
All these forms of attachment must also be interpreted as having
importance for preserving relative cell positions during active
movements of the animal. The chemical or molecular organization
of the cell surfaces is certainly not yet known in suflicient detail to
permit conclusions about the actual mechanism of attachment either
between adjacent cells or between cells and mesoglea. I believe,
however, that additional information will be obtained through
further studies using techniques for dissociating cells and by using

RICHARD L. WOOD 63

specific enzyme digestion. Analysis of appropriately treated material
with high resolution electron microscopy may provide further
information not only on the mechanism of intercellular attachment
but also on the molecular structure of cell membranes themselves.

Fig. 8. Interepithelial connection across mesoglea of Pelmatohydra. The
epidermal process (top) abuts against a gastrodermal muscle fiber. Note the
collapse of the normal intercellular separation at the region of contact
(arrow). ME, mesoglea. 13,000X.

64 THE BIOLOGY OF HYDRA : 1961

REFERENCES

1. Bennett, H. S., and J. H. Luft. 1959. s-Collidine as a basis for buffering fixa-

tives. ]. Biophys^ic. and Biochem. Cijtol. 6: 113-114.

2. Chapman, G., and L. Tilney. 1959. Cytological studies of the nematocysts of

Hydra. I. Desmonemes, isorhizas, cnidocils and supporting structures.
II. The stenoteles. /. Biophtjsic. and Biochem. Cijtol. 5: 69-84.

3. Hadzi, J. 1909. Ueber das Nervensystem von Hydra. Arb. zool. Inst. Wien. 17:

225-268.

4. Hess, A., A. Cohen and E. Robson. 1957. Observations on the structure of

hydra as seen with the electron and Hght microscopes. Quart. J. Micr.
Sc. 98: 31.5-326.

5. Luft, J. 1961. Improvements in epoxy resin embedding methods. ]. Biopliysic.

arid Biochem. Cytol. 9: 409-414.

6. Palade, G. 1952. A study of fixation for electron microscopy. /. £.v/;. Med. 95:

285-297.

7. Richardson, K. C, L. Jarett and E. H. Finke. 1960. Embedding in epoxy resins

for ultrathin sectioning in electron microscopy. Stain Technology 35:
313-323.

8. Robertson, J. D. 1959. New observations on the ultrastructure of the membranes

of frog peripheral nerve fibers. /. Biophysic. and Biochem. Cytol. 3:
1043-1047.

9. ScHAFFER, J. 1920. Vorlesungen iiher Histologic iind Histogenese. W. Engle-

mann, Leipzig, pp. 69-100.

10. Sjostrand, F., and E. Andersson. 1954. Electron microscopy of the intercalated

discs of cardiac muscle tissue. Experientia 10: 369-370.

11. Slautterback, D., and D. W. Fawcett. 1959. The development of cnidoblasts

of Hydra. An electron microscope study of differentiation. /. Biophtjsic.
and Biochem. Cytol. 5: 441-452.

12. Wood, R. 1959. Intercellular attachment in the epithelium of Hydra as

revealed by electron microscopy. /. Biophysic. and Biochem. Cytol. 6:
343-352.

DISCUSSION

WAINWRIGHT: Do you have any ideas concerning the site of
synthesis of the mesoglea?

WOOD: In Hyman and other textbooks it is claimed that the
mesoglea comes from both epithelial layers. I really have little more
to add. It is always strictly extracellular and has no hmiting mem-
brane, as Dr. Hess has already pointed out. It corresponds to the
connective tissue of higher forms. I don't know exactly how it
arises.

RICHARD L. WOOD 65

HESS: I've seen mesoglea in very young buds almost immediately
after their formation.

FAWCETT: I have no reason to regard the mesoglea as different
from any other epithelial basement membrane except for its greater
thickness. Where one has two epithelial or endothelial layers
arranged base-to-base in higher forms, one finds a layer of amor-
phous, PAS positive material which looks very much like a thin
mesoglea. I've always found this a very attractive prospect in
hydra research. Perhaps here is the best place to study the structure
and properties of basement membranes, and we might gain informa-
tion from the mesoglea that could be carried over to the basement
membranes which are such physiologically important structures in
higher forms.

I would ask you a question on terminology. I wonder why you
choose not to call these specialized zones of attachment "terminal
bars"? I certainly agree with you that "desmosomes" is preferable
from every point of view to the term "attachment plaques," but
isn't there an adequate functional reason for making a distinction
between desmosomes and those devices that occur next to the free
surface extending for the full length of the cell boundary and
which may very well have the function of preventing access of
material to the intercellular space. Isn't it desirable to distinguish
these elongated structures from the desmosomes which are circular
plaques that occur at many points along the confronted surfaces of
the epithelial cells and seem to be solely for attachment?

WOOD: I agree, Dr. Fawcett, but in my own terminology I
regard the term desmosomes as a more general term. I then say
this is a "terminal bar" type of desmosome. I'm sorry I didn't make
it clearer in my presentation. This concept of the generality of the
term desmosome comes from Schalfer's original description. I think
"Schussleisten," which, of course, was the terminal bar, is an earlier
term. Schalfer regarded the terminal bar as possibly arising from
fusion of a series of small plaques. I've used the term desmosome in
this general sense. I don't feel rigid about it, however.

HESS: We all try to get hydra fixed in an extended state. Some of
the things we see in the mesoglea might be very different, I think,
depending on the state of contraction of the hydra.

66 THE BIOLOGY OF HYDRA : 1961

LUNGER: I have electron micrographs of Campamilaria endo-
derm showing "terminal bar" desmosomes similar to those demon-
strated for hydra by Dr. Wood.

WOOD: This has been observed in several other forms. They
appear in planaria, and one is described briefly in Grimstone,
Home, Pantin and Robson's publication on Metridium.

SLAUTTERBACK: I'm willing to call these things "terminal
bars," but we must not put a functional significance upon this name
because we don't have any way of knowing that these structures are
excluding something from the epithelium and preventing it from
reaching the mesoglea. As far as I know the only obvious function
is attachment, is that right? To put it another way, we should not
apply the name terminal bars to the desmosomes of hydra because
the function of terminal bars, namely the impeding or preventing
the flow of water, electrolytes or other substances between cells,
has not yet been proved to exist in any organism or tissue other than
mammalian kidney.

WOOD: I think that the concept of terminal bar involves more
than just this concept of separating the lumen from the intercellular
space. It is a type of attachment which surrounds the entire surface
of the cell. In longitudinal sections it has a bar-like structure which
appears dense with certain types of stain. I agree that there is no
direct evidence that these specialized desmosomes of hydra func-
tion to prevent passage of water or other material intercellularly,
but I think that the idea is certainly reasonable because hydra is a
fresh water invertebrate and must osmoregulate somehow. There
is no kidney to do this and a reduction of exposed cell surface would
be one way to improve the situation.

FAWCETT: There is a piece of evidence not found in hydra
which indicates that terminal bars do have the function that has
long been attributed to them. In recent work on the proximal con-
voluted tubule of the mammalian nephron, Miller found that when
he administered hemoglol)in solution to mice, the hemoglobin that
filtered through the glomerulus and accumulated in the lumen of
the tubule is electron dense and served as a good contrast medium.
In electron micrographs one can follow the electron density of the
hemoglobin between the cells of the proximal tubule as far as the

RICHARD L. WOOD 67

terminal bar but no farther. Thus, at least for higher forms, large
molecules do not penetrate between cells and the traditional inter-
pretation of the terminal bar as a device for sealing the intercellular
spaces now has some experimental substantiation.

HESS: Some substances might even use those cross striations as
the steps of a ladder to climb into the hydra.

WOOD: I've thought of these cross connections as a system of
baffle plates that might slow down penetration between the cells.

Discussion on :

Is there a Nervous System

in Hydra?

HESS: Electron microscopists say that they can't see a nervous
system in hydra. And some of them say that a nervous system is
not needed to account for the movements of hydra because the
muscle cells in both ectoderm and endoderm contact each other
allowing muscle to muscle transmission to take place.

Dr. George Mackie from the Department of Zoology, Univer-
sity of Alberta, has a few slides showing some silver stains of the
nervous system. He might have something more convincing to
convey about the presence of nerve tissue in hydra.

MACKIE: This is a brief report on the results of a recent at-
tempt to stain ner\'es in the body wall of hydra and Cordylophora
using the classical Holmes silver technique. This work is still in its
initial stages. I will begin with CorcJi/lophora.

General topography of the nerve net. There is only one neuron-
system in Cordylophora, unlike Velella which has two histologi-
cally distinct plexuses. Neurons are abundant in the ectoderm of
tentacles (Fig. 2) and hydranth (Fig. 1). We have the following
figures for relative abundance of three tissue elements in a hydranth
preparation where all showed well:

Epithelio-muscular cells Neurons Cnidoblasts
231 94 64

Neurons also run in the ectoderm of the stem. In the hydranth the
neurons lie external to the muscle fiber sheet, running in the spaces
between the stems of the epithelio-muscular cells. They do not
follow the cell outlines, seen in surface view.

6.9

70 THE BIOLOGY OF HYDRA : 1961

Neuron types. Structurally there seems to be little difference
between neuro-sensory elements and purely nervous elements. Ap-
proximately one in eight neurons has a process running up to the
surface with a hair projecting externally (Fig. 3, 4, 8, 9), but of
those which are entirely sub-epithelial the majority have what seems
to be a reduced or rudimentary sense hair projecting into the sur-
rounding tissue space (Figs. 4, 7). It is possible that such cells
are modified sensory elements that have become or are becoming
transformed into neurons in the strict sense. However, this does
not exclude the possibility that they retain a sensory function, serv-
ing for instance to record deep touch or to give position sense.
The fibrous processes or neurites are similar in all these elements,
whether the cell has a hair or not.

Interconnections. The neurites associate freely, running side by
side for long or short stretches, but there is nothing to suggest that
they regularly form continuous connections. This nervous system
is quite unlike the closed system of Velella which shows every sign
of being a syncytium. It is much more like the non-syncytial open
system of Velella. The only evidence for continuous connections
is that here and there one finds binucleate neurons and in some
places there are suspicious-looking pairs of neurons which could
be the two halves of a binucleate pulled apart, but still in primary
connection. This gives me the opportunity to insert a remark
about the retention of primary connections between cells which
was discussed earlier, following the paper by Hess. Such connec-
tions have long been known in a variety of coelenterate cell types
including young cnidoblasts, interstitial cells and epithelio-muscular

Ectodermal nervous system of Cordylophora (Figs. 1-9) and hydra (Figs.
10-12) as seen in silver-stained whole mounts. Scale indicates 10 m/^.
Fig. 1. area of hydranth wall showing parts of five neurons; Fig. 2. neurons
in a tentacle; Fig. 3. neuro-sensory cell; Fig. 4. the three types of neurons;
Fig. 5. nerve fibers in contact with young cnidoblast; Fig. 6. bipolar ganglion
cell; Fig. 7. well-extended neurons in expanded epithelium; Fig. 8. neuro-
sensory cell showing root of hair in cytoplasm; Fig. 9. neuro-sensory ceil;
Fig. 10. neurosensory cell in contact with cnidoblast; Fig. 11. nerve fiber
tract: only two out of four fibers are in focus; Fig. 12. bipolar ganglion
cell.

Abbreviations: en. cnidoblast; g. ganglion cell; hs. subepithelial hair;
n. nerve fiber; p. process of neuro-sensory cell running to surface carrying
external hair.

2 ' "'

4^,

72 THE BIOLOGY OF HYDRA : 1961

cells. In cases where the intercellular bridge is long and slender and
still contains the relic of the mitotic spindle apparatus (Hirschler's
fusome) the structure may bear a strong resemblance to a nerve
fiber, especially in silver preparations where the fibers take the
stain like nerve fibers. I suspect that such fibers may have been
mistaken for nerves by certain workers.

"Innervation" of cnidohlasts. Given the abundance of neurites
and cnidoblasts it is not surprising to find frequent instances where
the two are in contact (Fig. 5). A rough estimate suggests that
about one in five cnidoblasts are in contact with part of a neuron or
neurosensory cell. No cases have been found where a neurite termi-
nates directly upon a cnidoblast such as Spangenberg and Ham
describe in H. litforalis. The contacts are apparently quite casual and
undifferentiated. Perhaps we should not speak of innervation until
we can show that these associations have functional significance.
Comparison of Cordijlophora and Hydra. Hydra has proved
harder to examine than Cordijlophora because the tissue is histologi-
cally denser and more elaborate. However, the silver preparations
do quite clearly show nerve elements. All that can be said at this
stage in the work is that the system appears generally similar to
that of Cordijlophora. Conventional neuro-sensory cells (Fig. 10)
such as Hadzi describes have been seen as well as subepithelial
ganglion cells (Fig. 12), some of which have a rudimentary hair
such as occurs in Cordijlophora. If there is a noteworthy difference
between the two forms it would seem to be the greater tendency
in hydra for neurites to run in bundles. This has been seen near
the hypostome, where bundles of up to four or five neurites (Fig.
11) have been followed for short distances, running around the ani-
mal in a circular direction. As to the connections, which many claim
to be continuous, I have nothing to say at the moment, except that
I have not seen any junctions which I would confidently interpret
as being continuous.

HESS: Does anyone else have any comments?

CLAY BROOK: 1 am very sorry that Dr. Spangenberg of the
Texas group was not able to attend this meeting to present her
studies of the nervous system in H. littoralis. I am afraid 1 cannot
do a very good job of describing her methods and conclusions.

DISCUSSION ON NERVOUS SYSTEM 73

Dr. Spangenberg used a methylene blue \'ital staining procedure,
with a neutral red counterstain, to demonstrate the nerve cells in
intact Hydra. I refer you to her recent publication (Spangenberg
and Ham, 1960, /. Exp. Zool. 14S, 195-202) for detailed descrip-
tions.

I obser\ ed many of Dr. Spangenberg's methylene blue prepara-
tions under phase contrast, and can report that they compare very
closely to Dr. Mackie's silver preparations. Nerve cells with from
one to seven fibrous processes were observed with interconnecting
fibers between many cells. While a complete nerve net could not be
stained all at once in any one animal, ner\'e networks in all regions
of the bod)- were seen in various specimens.

As Dr. Mackie reported, cnidoblasts are often found in close
contact with ner\'e cells. This doesn't indicate necessarily that there
is innervation of the cnidoblast, but the frequency of coincidence
is suggestive of that.

Dr. Spangenberg also identified multi-polar cells with the dis-
tinct morphology of neurons in Hydra preparations dissociated
into single cells with Hertwig-Schneider fluid. 1 think there is little
doubt that nerve cells and a nerve net do exist in Hydra.

HESS: If one wanted to be skeptical, it might be said that the
"nerves" that the Texas group shows associated with the cnidoblasts
are the discharged tubes of nematocysts.

BURNETT: I have recently received some photographs from
Semal Van-Gansen at the University of Brussels. She has dissected
out nerve elements from hydra with a fine needle. In the epider-
mis she finds the typical nerve net described by Hadzi (Fig. 1). In
the gastrodermis she does not find a net. Instead she finds a more
sparse distribution of nerve cells which do not resemble the small

Fig. 1. Isolated epidermal nerve cell (Semal Van-Gansen)

74 THE BIOLOGY OF HYDRA : 1961

bi-polar and tri-polar neurons of the epidermis. Those in the gastro-
dermis possess extremely long proeesses which branch profusely
(Fig. 2). She has suggested to me that perhaps the epidemial
net serves to coordinate the fast contraction of the longitudinal
fibers, and the neurons in the gastrodermis control the slower
contracting circular muscle fibers. She has been able to find sensory
cells both in the epidermis and gastrodermis. I have been able to
consistently demonstrate an epidermal nerve net by simply fixing
a whole hydra for Yi hour in 100% alcohol and then staining for a
few minutes in 0.1"? methylene blue. The nerve set is especially clear
in the transparent areas of the tentacles and peduncle. If this
interlacing network of bi-polar and tri-polar cells is not a nervous
system then morphologically it is a unique system in the animal
kingdom and one that must be reckoned with. Personally, I
feel certain it is a nerve net.

Fig. 2. Isolated gastrodermal nerve cell (Semal Van-Gansen).

HESS: Couldn't these "nerve cells and fibers" be cell membranes
radiating out from the intercellular spaces? Do the intercellular
spaces stain? This is a dissection, is it not?

BURNETT: Yes, this is a dissection.

HESS: Well, the cell membranes could be left intact radiating
from intercellular spaces filled with extensions of mesogleal sub-
stance. Impregnation of these elements could yield a picture appear-
ing like nerve cells and fibers.

DISCUSSION ON NERVOUS SYSTEM 75

SLAUTTERBACK: Before the argument is lost Iw default Td
like to inject a little bit of skepticism. I have no way of proving that
the nervous system does not exist, in fact, I am not sure that I
really doubt it. ( I was expecting Dr. Fawcett to stand up ahead of
me and say this.) But I would like to say that most of us who have
hunted for nerve cells with the electron microscope have been un-
able to find any. It is at least possible that this is because the
morphology of invertebrate nerves or hydra nerves is not readily
recognizable. But tliis is disturbing in view of the fact that there
are clear morphological criteria for the identification of nerves in
vertebrate tissues; they are readily recognizable with the elec-
tron microscope. In fact, I'd say more easily identified than in the
light microscope. Then too, it seems to me that the musculo-epitheli-
al cells are so beautifully organized for conduction in hydra, that
we don't really have to postulate the existence of a nervous system
which we can't see in order to account for the behavior pattern. I
recognize that it will probably take arguments more cogent than
these to refute a concept which has delighted liiologists for at
least 70 years. I have only to say that we can't see a nervous
system. We'd like things a little more sure.

HESS: Muscle to muscle connections, of course, are present e\'en
in mammalian smooth muscle. It wouldn't be an impossible situa-
tion for hydra to use muscle to muscle transmission to execute its
movements.

PASSANO: I doubt that this answers our discussion, l)ut it
might be of interest to tell this group of our success in recording
action potentials from hydra. A few years ago C. B. McCullough
and I tried to find out whether or not hydra showed non-decre-
mental through conduction by looking for ner\e action potentials.
We attempted to pick up actix ity of indixidual neurons, but what
we got, probably, were near-simultaneous action potentials from
several contiguous cells.

We had results with two types of preparations. The tentacle-
hypostome preparation (we cut off and discarded the column just
below the tentacular base) was threaded on a silver rod through
the mouth. In addition to serving to immobilize the animal the
rod served as a neutral electrode. While observing with a water

76 THE BIOLOGY OF HYDRA : 1961

immersion objective we brought the tip of a conventional capillary
microelectrode close to the cell body of one of the bipolar cells
miderlying the epidermis between the tentackilar bases. Occasional-
ly we picked up fairly strong, slow spikes, lasting 20 to 50 millisec-
onds and somewhat various in shape. They were always
associated with strong tentackilar contractions and always clearly
came before any movement was discernable in the area under ob-
servation. The tentacular reaction to glutathione did not elicit action
potentials, however.

The other successful preparation also gave action potentials
associated with strong muscle contractions. Here we used an intact
hydra suspended from the surface film and surrounded by a
wire ring to immobilize the animal and to be the indifferent elec-
trode. The microelectrode picked up action potentials after pene-
trating the basal disk, when the gastrodermal longitudinal muscles
contracted.

We believe that these electrical changes associated with either
tentacular or column "quick withdrawal" responses were nerve
action potentials, not muscle action potentials, since they came
well prior to muscle contraction, only with the quick, coordinated
contractions of all the muscle fibers, and since we only picked them
up sporadically.

HESS: From a nerve cell? Can you get your electrode inside a
nerve cell of hydra?

PASSANO: We think that they are from nerve cells, since we
attempted to place our recording electrode in the small bipolar
cells that underlie the epidermis. Since we did not have direct
coupled amplifiers available, we are not able to say whether or
not we ever penetrated nerve cells. Frankly, I doubt it.

Nematocyst Development'

David B. Slautterback

Depai'tmcnt of Anafonuj, The University of Wisconsin, Madison, Wisconsin.

To a cytologist one of the most intriguing aspects of the nema-
tocyst is that it is a secretory product hke many another, but unhke
those commonly studied, it possesses a very high order of structural
detail. To my knowledge, there are few rivals in this respect,
among them being the protozoan trichocyst which serves to remind
us that the coelenterates are not the only group with such highly
organized secretory products. Though understanding it not at
all, we are accustomed to the extremely intricate structures which
cells, in an enviable demonstration of community effort, can con-
struct in the extracellular space, such as hair and teeth. Still more
commonplace, and seemingly more intelligible, are intracellular
deposits of crystalline material. It does not stretch our imagination
seriously to conceive of the mechanism which brings about this
level of organization, impressive though it may be; for we can
produce this same or similar structure in the laboratory without
the inter\ention of cells. But comprehension of the mechanisms
involved in the intracellular elaboration of such a highly organized
body as the nematocyst challenges the best of our imaginative ca-
pacities. Speaking for the cytologist, the rewards are well worth
whatever effort is required for we can reasonably anticipate even
more than elucidation of this one mechanism common to a single
group of animals. Certainly new and better understanding of the
organelles with which all cells must work will ensue. This after-
noon we will hear several approaches to the understanding of
nematocysts, their production, structure and functions. For my
part I shall make a rather free interpretation of my assigned

^The work reported here was done during the tenure of U.S. Pul^lic Health Researcli
Grants RG5651 and RG6934.

78 THE BIOLOGY OF HYDRA : 1961

topic, devoting most of my time to one of the lines of differentiation
available to interstitial cells— the cnidoblast. Since Dr. Hess has
shown you excellent low power electron micrographs for orientation
I shall not include them in my presentation.

The small, relatively undifferentiated interstitial cell is found in
the gastroderm where it gives rise (at least) to the zymogenic and
mucous cells, and in the ectoderm where it may differentiate
into cells of the gonads, cnidoblasts and possibly some others. Figure

1 is an electron micrograph of a pair of interstitial cells in the
ectoderm of hydra. The nucleus is large and the nucleolus very
dense, but undoubtedly the most impressive feature of these cells
is the large number of cytoplasmic granules which are molecules of
ribonucleoprotein (RNP). In the cytoplasm of these cells, aside
from the ribonucleoprotein granules, or ribosomes, as they are
known to biochemists, there are no elements of the endoplasmic
reticulum, or at least they are very sparse. The Golgi complex is
represented, but only by a very few vesicles, showing a low degree
of organization. Another pair of interstitial cells is seen in Figures

2 and 3. They illustrate the fact that the nuclear membrane of
these cells has a specialization common to many other cell types,
as at "Po" in the figure. These small circles which appear in a tangen-
tial view of the nuclear membrane, and in longitudinal sections
as indicated by the arrows, represent what have been called nu-
clear pores. Whether or not they are physiologically "pores" or
"holes" in the membrane, I think remains unproved. But in any
case, it is likely that they represent specialized areas for transmis-
sion of materials from nucleus to cytoplasm. This is exactly the
kind of thing one would like to see in a cell which is about to differ-
entiate, or for that matter, in a cell which is undergoing rapid
mitotic division. However, the great desire to believe in such
things, does not really substantiate their functional significance. So,
while they may represent the lines of communication along which
the nucleus tells the cytoplasm "now it's time to divide," or "now
it's time to differentiate," this is largely speculative.

These pores may be seen to better advantage in Figure 4, where a
rather large piece of nuclear membrane has been cut in tangential
section. The abundance of these structures in the nuclear envelope
can be seen clearly.

DAVID B. SLAUTTERBACK 79

Another pair of interstitial cells is illustrated in Figure 5. These
show the same complex; the absence of endoplasmic reticulum
membranes and now an intercellular bridge (mentioned earlier
today) which shows a distinct confluence of cytoplasm between
the conjoined cells. And as usual, there is an accumulation in the
extracellular space of small dense particles. They measure about
250 to 300 Angstroms and in all respects resemble the particulate
glycogen described by Fawcett and Selby in the atrial muscle of
turtle heart and by now in numerous other cell types. I should point
out, however, that it is not very common to find glycogen particles
extracellularly except here in the ectoderm of hydra. And in these
cells, glycogen, in my experience, as particulate glycogen, has never
been demonstrated intracellularly. Never within the interstitial cells
nor developing cnidoblast; only extracellularly. This would fit well
with the suggestion that glycogen is broken down at the cell
membrane.

Returning to the intercellular bridges, your attention is direct-
ed to its thickened membrane which seems to impart enough rigidity
to the structure to resist deformation by the frequent shape changes
of the animal as a whole. The plasmalemma is continuous from
one cell to the other through the tubular bridge, although it is
sharply reflected upon itself twice, and bears a peculiar annular
expansion midway along the length of the bridge. Figure 6 is a
striking demonstration of this form and the continuity of cytoplasm
between the two cells. The vesicles in the center of the bridge
could hardly be said to belong to either one cell or the other. Prob-
ably the most important function of the intracellular bridge is to
synchronize differentiation and thus provide large numbers of
cnidoblasts in the same stage— reaching maturity at the same time.
But, also in the early stages of cnidoblast development, when the cell
is primarily concerned with proliferation, these intercellular bridges
undoubtedly serve to synchronize the mitoses. It is possible, with
some speculative stretch of the mind to suppose that the sub-
stance which synchronizes these mitoses must therefore be a
soluble substance, readily and rapidly transmitted from one cell to
the other. And, so we have here some evidence for the fact that the
nucleus when telling cytoplasm to begin a mitotic division,
transmits this information by some relatively small molecule, or

80 THE BIOLOGY OF HYDRA : 1961

at least a rapidly diffusible one which quickly can reach an equili-
brium level within the group of developing cnidoblasts. In my ex-
perience these are usually 14 to 18 cells joined in a cluster,
from which it is evident that a rapid diffusion rate is necessary to
keep them all very closely synchronized.

This synchrony is illustrated by the pair of interstitial cells in
Figure 7. The dense clumps of granules are the chromatin material,
and only remnants of the ruptured nuclear membrane persist.
These are not two daughter cells in anaphase, they are in late
prophase, so the mitoses are quite closely synchronized. When these
cells divide for the last time, the diplosome remains near the
plasmalemma ( Fig. 8 ) . The remnants of the achromatic figure, the
spindle fibers can be seen clearly (S). They appear to be thin
tubular structures on the order of 200 Angstrom units in diame-
ter. Whether or not these spindle fibers have any progeny, or any
remnant left in the fully differentiated cnidoblast, cannot yet be
said. The possibility exists, and I shall point out at a later time what
I believe to be their fate.

You will see at "G" in the figure, a large number of vesicles
belonging to the Golgi complex. Most of them do not have ribo-
nucleo-protein granules upon their surfaces; but some do and still
others have granules on one side and none on the other which may
be interpreted as supporting the arguments for the continuity be-
tween the endoplasm reticulum and the Golgi complex.

Dr. Fawcett pointed out earlier today that some groups of
cells are not joined together by intercellular bridges of the very
intricate structure that you have just seen, but rather by simple dis-
continuities of the membranes, an example of which appears in
Figure 9. It is difficult indeed to argue that these are not artifacts
of preparation techniques. But one can only say that they are fre-
quently seen, and they appear in cells which otherwise seem very
well preserved. However, two of the cells in the micrograph are
bound together by an intercellular bridge of the specific type, and,
it is not at all uncommon to see both types of continuit}^ within
the same cluster. In fact, joining the same two cells together.

Now when the endoplasmic reticulum begins to appear, we
see coincidentally the first appearance of the nematocyst. The
reticulum first appears as scattered vesicles in the cytoplasm,

DAVID B. SLAUTTERBACK 81

rather spherical in appearance (Fig. 10); they have a very low
density content. You can see at the arrows, for instance, a small
amount of material within those vesicles. The nematocysts are indi-
cated by "Ne"; one in the upper left hand corner and one in the
lower right hand corner. It is quite difficult to detemiine exactly
which name belongs with which nematocyst. But I would like to
say by way of record here, that within one cluster all of the nemato-
cysts we have seen are definitely of the same type and they continue
to be the same type throughout the stages of differentiation. The
relatively homogeneous area is the capsule of the nematocyst, and
the granular area will become the tube. Around the open end,
where the operculum will finally appear, there is a very dense ag-
gregation of smooth vesicles which clearly belong to the Golgi
Complex "GC." Notice again, the presence of glycogen granules
between cells.

In Figure 11 there is a cluster of cnidoblasts, early in their
differentiation, and you see several sections of nematocysts and
the nuclei of these cells. The intercellular bridges are quite conspicu-
ous and now the endoplasmic reticulum has become considerably
more prominent. The latter is seen mostly as sections of tubular
structures, but there is some tendency to form flattened cysternae,
typical of such secretory cells as the pancreatic acinar cell, for
example. This section, however, has missed the Golgi zones.
This particular illustration serves p r i m a r i 1 y to point out the
remaining cytoplasmic bridges, and the progressive increase in
vesicles of the endoplasmic reticulum. Figure 12 is a higher magni-
fication view of cells at a slightly more advanced stage to emphasize
the persistence of the intercellular bridges and the continuity of
organelles, not simply continuity of cytoplasmic matrix, but or-
ganelles seem to be shared between the cells.

As the nematocyst develops, it acquires the appearance in Fig-
ure 13. The Golgi complex is becoming very much more abundant.
The centrioles, which are really a diplosome, remain at the open
end of the forming capsule. The capsule is the lighter amorphous
or faintly fibrous part, and the darker granular material is the
forming tube protruding from the open opercular end of the cap-
sule. Notice that the Golgi complex forms a close-fitting cap over
the growing end of the tube. There is a continuous membrane sur-

82 THE BIOLOGY OF HYDRA : 1961

rounding this forming nematocyst which is agranular, and in all
respects resembles that of the Golgi complex. You will see that the
Golgi complex is formed as usual in vertebrates of flattened vesicles,
expanded vesicles, and small spherical ones. It has been said that
such appearances are not common in invertebrates and represent
more of a vertebrate type of Golgi complex, so then hydra cnido-
blasts have a vertebrate type of Golgi complex, if that's the case.
The large body here at the top of the figure is lipid droplet, and in
our experience lipid droplets are a ubiquitous finding in all secretory
cells. Of course, lipid droplets are found in virtually all cell types,
but a relatively sudden accumulation of lipid seems to go hand in
hand with the differentiation of these secretory cells.

In another section of the opercular end of a developing nemato-
cyst ( Fig. 14 ) the Golgi membranes surrounding the growing tubule
can be seen more clearly. In the Golgi zone, the three types of
vesicles are evident and especially prominent in this micrograph,
is this large expanded one (indicated by an arrow) whose
contents appear every similar to those of the nematocyst capsule.
The only appreciable difference seems to be a slightly greater den-
sity of the material in the nematocyst than in the Golgi vesicle.
One can often see areas where these Golgi vesicles seem to increase
gradually in size getting larger and larger, and finally one of the
vesicles seems to join by fusion of its membrane with that of the
membrane surrounding the nematocyst tube or rod. This process
is illustrated in Figure 15. It bears a remarkable resemblance to the
mode of release of secretory granules in other cells in which the Gol-
gi membrane surrounding the granule fuses with the plasmalemma
and the membrane is broken at the point of fusion releasing the
secretory product and adding the Golgi membrane to the plasma-
lemma. As you can see the endoplasmic reticulum is continuing to
develop. We are not yet past the peak of protein synthesis in this
cell. That similar configurations are present in the isorhizas is
evident from Figure 16. Here a large Golgi vesicle is being added
to the nematocyst tube. Though some degree of uncertainty re-
mains as to the identity of these developing nematocysts, those
which you have seen before were probably desmosomes, but this
one is an isorhiza, though whether it should be regarded as holo-
trichous or atrichous, I cannot say. But again, you see the cen-

DAVID B. SLAUTTERBACK 83

trioles at the opercular end, and the Golgi complex aggregated
around the open end of the nematocyst.

Figure 17 illustrates a very recent observation in our laboratory.
The micrograph shows a cross section of the neck region of a devel-
oping nematocyst. The accumulation of vesicles of the Golgi complex
indicates the forming tul:>e has not yet extended very far out of
the capsule. Immediately surrounding the Golgi membrane, which
encases the nematocyst, is a row of very small tubules. They are
about 180 A in diameter with a lumen about 75-80 A in diameter
and a wall thickness of al)Out 50 A. In the upper right quadrant of
the figure they are seen in perfect cross section. The function of
these elements is not yet clear, but some of their structural relation-
ships may be significant. In the interstitial cells they are found in
groups scattered through the cytoplasm. Within the groups tubules
are arranged at right angles to each other. They are evidently contin-
uous with the tubules which have been interpreted as spindle fibers
in Figure 8. In intermediate stages they are as figured here and in
later stages (as Fig. 27) they continue to surround the nematocyst,
oriented parallel to its long axis and are continuous at one end
with the rootlets of the stiff rods ( described later in this paper ) and
at the other with dense coils of tubules in the nuclear zone and seen
as fibrous bodies in Figure 27. The only suggestion of function
is seen in the relationship at the arrow in Figure 17. Here one
tubule appears to be in direct communication with one of the small
spherical vesicles of the Golgi complex. Whether this indicates a
separate mechanism for the production of nematocyt capsule is not
yet clear.

Now to return \ er\' briefly to the endoplasmic reticulum. Figure
18 shows a fairly earh' group of cells with small tubular elements
of the reticulum. In Figure 19 you will see a fairly late stage in the
development of the cnidoblast. The cell has about reached the peak
of its synthetic activity, and the endoplasmic reticulum now assumes
a more packed formation and you see many flattened sacs which are
disposed in a concentric array around the nucleus. The wider spaces
(also marked with a star in Fig. 20) are areas where the reticulum
has been cut obliquely and are not in reality such wide diameter
structures. And finally the condition illustrated in Figure 20 is
reached when the reticulum fills most of the cell. During the fomia-

84 THE BIOLOGY OF HYDRA : 1961

tion of the nematocyst, the Golgi complex is at all times in close
proximity to the tip of the forming tube and that tube is formed
out in the cytoplasm. It may become very very long and coiled
around through the cytoplasm, but the Golgi complex caps the
growing tip.

In Figure 21 is a cnidoblast which has passed its peak of syn-
thetic activity. We considered for sometime that the expansion
of these endoplasmic reticulum vesicles was a fixation artifact due to
osmotic differences in the fixative as compared to those within the
cell. But by using a very wide variety of osmotic strengths and
hydrogen ion concentrations, we have convinced ourselves that
this is exactly what happens to the reticulum after it has passed
the peak of synthetic activity. It begins to swell up, perhaps with
an acute hydration of its contents. I wouldn't like to extend myself
on that point, but in any case they do become vesicular again.

In Figure 22 you will see a nematocyst, which shows how this
forming tubule continues around through the cytoplasm. The ab-
sence of Golgi vesicles from this section clearly indicates that there
are still more coils of tubule elsewhere in this cell for if the tip were
here we would see the Golgi membranes surrounding it.

The cell in Figure 23 shows a still more advanced condition
and this one is an early stenotele. The Golgi membrane sur-
rounding the nematocyst is clearly discernable, and now we begin to
see a concentration or aggregation of dense granules which were
once randomly distributed. It is in this zone that the spines and
thorns of the nematocyst tube will be formed. In this micrograph
there are four sections through the coiled tube which is still outside
of the nematocyst capsule. The darker bodies are mitochondria, and
the endoplasmic reticulum is clearly vesicular and considerably de-
creased in amount indicating the end of the synthetic phase. Though
not present in this illustration, the Golgi complex is still active evi-
dently collecting and concentrating material synthesized earlier in
the now regressing reticulum.

A more advanced stenotele cut longitudinaly is seen in Figure
24. The tubule has been withdrawn and the open end of the
capsule is closed by the operculum. The laminated structure of the
operculum can be seen in Figure 31. The arrows point to the ele-
ments which were originally distributed at random throughout the

DAVID B. SLAUTTERBACK 85

substance of the forming tube, and have now just begun to form
the tubular wall and the spines and thorns. So this, I am sorry
to say, is the stage soon after the retraction of the tube, which was
as you saw before, wound throughout the cytoplasm. And I presume
that this retraction is a very rapid process because we have never
seen (or recognized) it in progress. On the other hand it may be
that some of the tubes which we see lying coiled out in the cyto-
plasm having a cross section somewhat thicker than usual, are these
tubes undergoing withdrawal. In any case it is clear that there is no
visible structure in the tube before it has been withdrawn into the
capsule and that all of the intricate structures which appear later on
are formed without immediate contact with cytoplasmic organelles
and the mechanism of this astonishing feat remains an enigma.

Figure 25 illustrates some of the elaborate detail of the structure
of a stenotele and points out that the endoplasmic reticulum, which
has reached a vesicular stage, is now disappearing and that the
phospholipids of that membrane have gone some place else. It
might be interesting to follow the displacement of these phospho-
lipids with histochemical procedures, but we have not as yet tried
such things.

The isorhiza in Figure 26 illustrates a similar course of events
in that type of nematocyst: the endoplasmic reticulum has become
vesicular and vanished to a very large degree. The coiled tube is in-
dicated at "T," and I presume that this is a holotrichous isorhiza be-
cause, in some areas (arrows), we see what appear to be develop-
ing thorns. At "Cn" in the upper right of the figure is the region
where before we saw the diplosome and now we see what Hyman
has referred to as the stiff rods which surround the operculum, a
part of the cnidocil appartus.

A similar degree of differentiation is seen in Figure 27, but the
section has passed through the operculum and the cnidocil. One
of the centroiles of the basal granule is at the base of the cilium,
which, I believe, is just in the process of forming, and is quite broad
in diameter. And just outside it, you can see one of the stiff rods.
Now this is not the dense part, which you saw in the section im-
mediately preceding, but this is the part which corresponds to the
body of the cilium itself. The endoplasmic reticulum is much dimin-
ished. The Golgi complex has retreated to the basal area of the cell

86 THE BIOLOGY OF HYDRA : 1961

and has often been described here, by silver stains, as a dense body
in the basal part of the cnidoblast, but I believe it is simply an inac-
tive Golgi complex. Immediately below it are very fine filaments
which by newer techniques appear to be fine tubules (see Fig. 17).

Figure 28 is a fortuitous section through a stenotele which is
fully developed. The parts of the nematocyst are readily recog-
nizable including the operculum (O), two of the three spines,
and the faintly striated tubule. The membrane surrounding
this structure is quite obvious. The cilium with its basal granule and
one of the "stiff rods" are also prominent. Now I think it's obvious
that this so-called stiff rod is very similar to the cilium in structure,
but you can see faint longitudinal striations in the cilium which are
absent in the stiff rod. Another structure which appears often in this
zone is the multivesicular ( M V ) body which most closely resembles
the lysosomes of DeDuve. I should also like to point out that there
are very fine filaments visible in this micrograph which are attached
to the cilium and to the stiff rods; in more favorable sections they
also appear to be attached to the circumference of the operculum,
and may serve in the mechanism of firing the nematocyst.

In Figure 29 is a cross section of a stenotele. In the center of
the micrograph the three heavy spines of the base of the tube can
be seen; a dense material is gradually accumulating in them from
the periphery inward. The peculiarly convoluted material aromid
the spines is the base of the tubule itself and the conspicuous cross
striation of it has a repeat period of about 150 A; that is, each
light line measures about 75 A wide as does each dark line. The fact
that this same periodicity is seen in longitudinal section (Fig. 30)
suggests that the tubule is composed of a crystalline array of rod
shaped molecules. ( I am not able to explain the difference between
my measurements and those of Dr. Chapman though it is not impos-
sible that they vary with degree of development or dehydration.)

I am not going to deal extensively with the cnidocil structure
at this time, but I would like to make a few additional observa-
tions. In Figure 31 you will see that the stiff rods of the cnidocil
appear first as a straight row of dense bodies connected by a fine
dense line. And at one end of that row of bodies, there is a basal
granule of an unmodified cilium (not visible in this section). This
cilium can be seen in figure 32 where the stiff rods, now quite well

DAVID B. SLAUTTERBACK 87

developed have begun to form a circle around the operculum.
Notice that it is surrounded by fine filamentous structures which
show a repeat period somewhat larger than 300 A. This section is
slightly oblique to the plane of the rods so that in the upper left
it has passed through the modified ciliary part and in the center
and to the right has passed through the cross-striated rootlet. There
are 21 of these plus the true cilium.

In the next illustration you can see the relationship between
the rootlet-like structure and the ciliary-like structure of the stiff
rod. If you follow the membrane around the ciliary x)art you
see that it passes below and peripheral to the upper end of the
clearly cross-striated rootlet. It is remarkable that the rootlet which
in the ordinary cilium is supposed to lend structural and function-
al stability, should be offset in this way. Though there is little evi-
dence to support the notion at this time, such an arrangement
might function as a hinge with the ciliary part bending outward
and the rootlet remaining fixed.''

Figure 34 shows a slightly oblique section through the complet-
ed apparatus. Notice the 21 rods and the eccentrically placed
cilium. Again the fine filamentous material which interconnects all
parts of the apparatus and the operculum.

The last two micrographs (Figs. 35 and 36) are taken from a
section of a very different animal, but I want to use them to illus-
trate an important consideration about the functioning of the
endoplasmic reticulum in the cnidoblast. It is not evident from the
developing cnidoblast that the ril^onucleoprotein granules must be
or even can be attached to a membrane of the endoplasmic
reticulum in order to function in the synthesis of new protein. It
has been argued for some time that only the free granules of ribonu-
cleoprotein are active and that after synthesis is completed the free
granules move with their product to the endoplasmic reticulum
where the product is separated and added to the contents of the
lumen of the vesicle. We have seen in the proliferating inter-
stitial cells that free granules arc active in the production of protein
"for domestic consumption," i.e. new protoplasm. In the case at
hand we have a secretory cell in the gut of a small earthworm.

•"'It should be pointed out that the tubules illustrated in Figures 8 and 17 appear
to be continuous with the rootlets of the stiff rods.

88 THE BIOLOCY OF HYDRA : 1961

Enchytraeus fmgmcntosus (Fig. 35). This cell is a very active
protein secretor and this is the peak of its synthetic activity. It has
become completely filled with endoplasmic reticulum plus a few
secretory droplets and a very few mitochondria. In Figure 36 I
think I can convince you that there are no free ribonucleoprotein
granules in this cell; thus, attached RNP granules induce synthesis
of protein for export from the cell; whether or not free ones do, I
cannot say.

(I cannot distribute the responsibility for the interpretations
presented here, but I would like to acknowledge the important con-
tribution of Prof. Don W. Fawcett to this work. )

Figures 18, 23 and 15 are reprinted here by courtesy of the
Journal of Biophysical and Biochemical Cytology. They appeared
in volume 3, page 441 of that Journal.

The following abbreviations have been used in the accompanying
illustrations:

Centriole — Ce Nucleus — N

Cnidocil Apparatus — Cn Nuclear Envelope — Np

Endoplasmic Reticulum — ER Nuclear Pores — Po

Golgi Complex — GC Nucleolus — No

Intercellular Bridge — B Operculum — 0

Lipid Droplet — L Particulate Glycogen — Gy

Mitochondrion — M Plasmalemma — P

Multivesicular Body — MV Ribonucleoprotein Granules — RNP

Nematocyst — Ne Spindle Fibers — S

Nematocyst Capsule — C Zymogen Droplet — Z

Nematocyst Tube — T

Fig. 1. A pair of interstitial cells showing granular cytoplasm. 8,900X.
Fig. 2. Nuclear pores in an interstitial ceil. 10,000X.

RNP

'mm

\ji

■> .^-Wiix..

Fig. 3. Same cell as Figure 2 somewhat enlarged. 13,000X.

Fig. 4. Tangential section of the nuclear envelope in an interstitial
cell showing the distribution of "nuclear pores." 32,000X.

--%

Fig. 5. A pair of interstitial ceils bound together by an intercellular
bridge. 12,500X.

Fig. 6. Enlargement of intercellular bridge similar to Figure 5. 22,000X.

Fig. 7. Two interstitial cells from a single cluster, both in late prophase.
9,500X.

Fig. 8. Diplosome of an interstitial cell with attached spindle fibers which
are in fact tubules. 29,000X.

Fig. 9. Two types of protoplasmic continuity in a cluster of interstitial
cells. 12,000X.

Fig. 10. Early cnidoblasts showing beginning development of endoplasmic
reticulum, Golgi complex and nematocyst coincidentally. 17,000X.

B '

' w-/^ i

<-'

SSI-

' ,.r **

•c..
^

''1

1 j^^tm&^S^^

fe'" 1% •

1 ;

Fig. 11. A cluster of cnidoblasts slightly more advanced than those in
Figure 10. 17,000X.

■K

^^1

-^ /

Fig. 12. An intercellular bridge in a pair of cnidoblasts slightly more
advanced than those in Figure 11. 22,000X.

Fig. 13. This longitudinal section of a nematocyst shows the cop-like
arrangement of the Golgi complex over the growing tip of the tubule.
15,000X.

100

.'W'*

>3-

101

Fig. 14. Similar to Figure 13, but somewhat enlarged. 19,000X.

Fig. 15. The membrane of a large Golgi vesicle has just fused to the mem-
brane surrounding the nematocyst and in this way added its content to the
previously synthesized tube material. 32,000X.

102

'^MOKaBkSxmsk ^i

lu^

Fig. 16. A cnidoblast containing a developing isorhiza. The same process
lustrated in Figure 15 is seen here. 23,000X.

Fig. 17. Cross section of the neck of a developing nematocyst. 46,000X.

104

t-'

,/■

■^1

,v.'

"^ '^'^«»«ta™SMiii

fjjjUh-T.

105

Fig. 18. A cluster of early cnidobiasts undergoing synchronous differen-
tiation. 13,500X.

Fig. 19. A cnidoblast approaching the peak of synthetic activity. 20,000X.

106

101

Fig. 20. In this cnidoblast protein synthesis is going on at the maximum
rate. 22,000X.

108

m'^^w<'mi

lOU

Fig. 21. This cell has passed the peak of synthetic activity and the reticu-
lum has begun to vesiculate. 8,900X.

Fig. 22. A considerable part of the coiled external tube has been cut in
longitudinal section. 15,000X.

110

y' .'t ,?

^.,

111

Fig. 23. Further regression of the endoplasmic reticulum is evident in
this cnidoblast, as are several sections of the coiled tube. 8,900X.

Fig. 24. The tube has been withdrawn into the capsule and the open end
closed by an operculum. Fine structure of the tube has begun to form.
21,000X.

112

2 3 4. . • %;

2 4

¥*^

00^'

113

Fig. 25. A nearly mature stenotele. Rupture of the capsule is artifactual.
n,500X.

Fig. 26. A longitudinal section of an isorhiza showing regression of the
endoplasmic reticulum and development of the cnidocil apparatus. 18,500X.

114

115

Fig. 27. This figure is similar to Figure 26 but the nematocyst is a sten-
otele. 10,500X.

Fig. 28. A stage simiior to that in Figure 27 but somewhat enlarged.
16,500X.

116

• 27

117

Fig. 29. Cross section of a stenotele tubule. 42,000X.

Fig. 30. Longitudinal section of a stenotele tubule. 30,000X.

118

30, .^ ".,

119

Fig. 31. This is the same cell as in Figure 24. Notice lamination of oper-
culum and straight row of dense bodies of early cnidocil. 44,000X.

Fig. 32. An oblique section through the opercular end of a stenotele
showing partial encirclement by the developing cnidocil. 29,000X.

120

' vm-ifes-.\ '\*V N>^ ""'

IV^

''^-' ^^!l*^ *._ '

■ M

* \ --y'

121

Fig. 33. Longitudinal section of a "stiff rod." 49,000X.

Fig. 34. Oblique section of a fully differentiated cnidocil apparatus.
32,000X.

122

-t

^y-^

■jr

...•fit ■' ...JXOt • .- -«^ ■ '•% •

e.^«&

^.: V^iil^

1<s.

■^•^

i2.-3

Fig. 35. This is a zymogenic cell from the gut of an earthworm, Enchy-
traeus fragmentosus. 24,000X.

124

li^

M M

125

126

THE BIOLOGY OF HYDRA : 1961

Fig. 36. An enlargement of a part of Figure 35. 41,000X.

DISCUSSION

HAND: Is it your impression that the tube forms outside the
capsule of the nematocyst?

SLAUTTERBACK: Yes. I think the fact that the Golgi complex
remains associated with the tip of the forming tubule makes it
difficult to imagine that this is a prematurely fired tube. Further-
more, it is difficult to imagine a situation where every time the tube
fired inside the cytoplasm it would end up with its tip in immediate
association with the Golgi complex.

ROSS: Isn't there a big volume change in the cnidoblast as it
develops?

SLAUTTERBACK: I haven't done any very accurate measure-

DAVID B. SLAUTTERBACK 127

ments, but I would say no, there is not. I think after it's fired there
seems to be quite an enlargement.

LOOMIS: Do you picture the tube starting to be made at the
mouth of the capsule, and then progressing out, smaller and
smaller?

SLAUTTERBACK: That's right, except that this capsule at
first is only a sort of crescentic shell or cup. It has not yet formed a
full flask-shape structure.

LOOMIS : The tip, then, is the last part made?

SLAUTTERBACK: Yes.

LOOMIS: Is there anything like a hypostome or ring of produc-
tion around the capsule lip, so that the tip would be the first part
made as in a tentacle?

SLAUTTERBACK: No sir, not at all. In fact, completely the
opposite.

LANE: Would you like to speculate about the mechanism of
withdrawal of this externally formed tubule?

SLAUTTERBACK: Maybe Dr. Fawcett would like to speculate
on that.

FAWCETT: No, I would not. I am content to regard it as the re-
verse of the mechanism of firing!

This is a minor point, but it may be of interest that the contents
of the nematocyst are not only highly diverse from one kind to the
other, but even the operculum is quite characteristic of the particu-
lar type of nematocyst. In this slide ( Fig. 1 ) is one quite different
from the one in Figure 2 and from any that Dr. Slautterback showed,
in that the operculum has an interesting laminated structure. I have
no idea as to what the significance of this lamination is.

HAND : Do you know what nematocyst that is that you showed on
the slide?

FAWCETT: No, I do not.

WOOD: Hasn't the presence of some type of an intracapsular at-

"^Ido-. ^,,jtai

"«4.

Fig. 1.

DAVID B. SLAUTTERBACK 129

tachment region at the opposite pole from the opercuhim been
described in the Hght microscope hterature? Wasn't this interpreted
as being important in retracting the tube after it had been formed?
I seem to recall something of this sort. I have seen electron micro-
graphs of nematocysts that show a specialized area at the opposite
end from the operculum. I wonder if you would comment on
this?

SLAUTTERBACK: Yes, I understand that such structures have
been described around the outside of the capsule. I have never
seen these.

HAND: Cutress described such a structure inside the microbasic
mastigophore in his paper on anthozoan nematocysts, but he did not
suggest that it was used to withdraw the thread.

SLAUTTERBACK: I have never seen anything inside that didn't
obviously belong to the tubule itself, and these would be everted
with it: spines, thorns, and things of that sort. George, did you want
to speak about that?

CHAPMAN: I was wondering if Dr. Wood was referring to the
plug in the basal capsule pore that Kepner and his colleagues de-
scribed.

SLAUTTERBACK: Of the capsule itself?

CHAPMAN: A dense mass of material which they originally
described as being a plug in the pore in the basal portion of the cap-
sule. The function of it is not understood. It was thought to be the
end of the spiral tube and to be converted to magma just prior to
discharge.

SLAUTTERBACK: Have you seen such a structure?

CHAPMAN: Yes, I think one of our pictures of a few years ago
presented it rather vaguely.

SLAUTTERBACK: I haven't seen it.

The Fine Structure of
the Stenoteles of Hydra'

George B. Chapman

Department of Anatomy, Cornell University Medical College, New York, N. Y

111 the more than two hundred years which have passed since
Leeuwenhoek ( 9 ) and Trembley ( 13 ) first referred to the nemato-
cysts of hydra, these structures have been the subject of a great
number of anatomical, biochemical and physiological investiga-
tions. As is usually the case, as many questions were raised as
were answered. Recently, in an attempt to answer some of these
questions, Hess et oJ. (6), Slautterback and Fawcett (12) and Chap-
man and Tilney (3, 4) studied ultrathin sections of nematocysts
in the electron microscope. It goes without saying that these studies
have contributed appreciably to our knowledge of nematocyst
structure, development and function. Incidentally, the studies have
reviewed rather thoroughly the extensive literature in this field.
This report, therefore, omits such a review and refers to the previ-
ous work only where it is pertinent to the discussion of the most
recent observations.

It is especially gratifying that the nematocyst, a structure worthy
of study solely on the basis of its morphology, should also provide
valuable information on cellular differentiation and synthesis, as
Slautterback and Fawcett (12) have so clearly shown. Thus, once
again, resort to the study of a classic animal has provided new data
—in this case, concerning the role of the Golgi apparatus and endo-
plasmic reticulum in the production, by the cnidoblast, of an
elaborate and highly specialized cell inclusion. Furthermore, evi-

^This work was supported l)v United States Public Health Services research tyrant
E-3517.

131

132 THE BIOLOGY OF HYDRA : 1961

deuce exists which suggests that the new findings are generally
applicable to problems of cell growth and cell differentiation.

The present report will be limited to some recent observations
pertaining to the structure of stenoteles, the largest of the four types
of nematocysts of Hydra.

MATERIALS AND METHODS

Entire Hydra (H. vulgaris and H. littoralis) were fixed for 1 to
2 hours at 2 to 5 in 1 per cent osmium tetroxide buffered to a
pH of 7.6 with acetate-veronal buffer. The fixative also contained
about 0.7 per cent sodium chloride and a few drops of 0.11 M
calcium chloride.

Following fixation, the specimens were washed, dehydrated
through a graded ethyl alcohol series and were embedded in Epon
according to a schedule developed in Dr. H. Stanley Bennett's
laboratory by Drs. J. H. Luft and R. L. Wood (1).

Sections were cut with a Servall Porter-Blum ultramicrotome
using glass knives prepared in the laboratory. The sections were
floated onto distilled water and were picked up on collodion-coated
grids (Fullam #2001). Staining with uranyl acetate for 1 to
2 hours was carried out according to the method devised by
Watson ( 14 ) . Electron microscopy was accomplished with an RCA
electron microscope, type EMU-2D, equipped with a 0.015 inch
externally centerable (Canalco) platinum condenser aperture and
a 50^ copper objective aperture in the standard objective pole
piece.

OBSERVATIONS AND DISCUSSION

The present studies of stenotele fine structure have revealed
several interesting new features. Figure 1, a sagittal section of a very
nearly mature stenotele, shows, in addition to the structures previ-
ously described by Chapman and Tilney (1959b), viz., capsule (C),
operculum (O), invaginated capsular wall (ICW), stylets (ST),
spines (S), matrix (M) and tubules (T), what appears to be a
most fortuitous section through the enlarged "head " of the tubule

GEORGE B. CHAPMAN 133

(HD). Distal to the head, may be seen a narrowed extension
of the tubule ending in a hook (HK). Just proximal to the
head, the longitudinal section reveals a rather fluted contortion sug-
gesting why the cross sections of tubules often appear as three-
bladed propellers. The enlarged head of the tubule is of particular
interest for it is the only non-structural portion of the capsular con-
tent which is distinct in its texture from the matrix. One could
speculate that this small packet of material might possibly rep-
resent the location of the venom of Hydra. It might further be
speculated that the minute (approximately 150 A in diameter)
dense granules in the tubule head represent the 5-hydroxy tryp-
tamine which Welsh and Moorhead ( 15 ) have suggested may be
a constituent of coelenterate venom. Such a location for the
venom would be most effective for the presence of the hook at the
termination of the tubule suggests that the head portion of the
tu]3ule may be attached to the base of the invaginated capsular wall
and thus be drawn out of the discharging nematocysts in advance
of the rest of the tul)ule, thus causing the head to encounter the
Hydras prey at the earliest possible moment, thereby facilitating
the utilization of the venom. If the enlarged head of the tubule
does not contain the \'enom, it is difficult to imagine where the
venom could reside save in the capsular matrix material. The lat-
ter possibility seems less satisfactory from an operational stand-
point. (The head of the tubule may be projected rapidly to a dis-
tance of many microns from the body of Hydra, while the matrix
material would have to depend largely on diffusion to reach the
prey. )

The above observations thus tend to reopen the question of the
nature of the tubule discharge mechanism. If eversion occurs, the
contents of the tubule head could encounter the prey only when
the tubule is fully everted. Prey located close to the surface of
Hydra might be by-passed by the head of the tubule and any
possible function of the contents wasted. If the head is discharged
somewhat like a fishing lure is cast, it could quickly encounter
prey located anywhere within its maximum range. The latter meth-
od of discharge, of course, raises the questions of how the head of
the tubule could get outside of the invaginated capsular wall and

134 THE BIOLOGY OF HYDRA : 1961

how the contents of the head could l^e released. It is hoped that
further study may clarify this intriguing situation.

Another structural feature of the stenotele not previously de-
scribed is the occurrence on the base of each spine of a bulbous
enlargement (arrow). This arrangement would seem to insure a
firmer attachment of the spines to the invaginated capsular wall.

Figure 2 also illustrates, in a transverse section through the
basal portion of a stenotele of a stage of maturity similar to that
of Figure 1, the bulbous enlargement (arrows) of the bases of the
spines. This figure is also interesting in that it includes a cross
section through the enlarged head of the tubule (HD).

Figure 3, a transverse section through the apical portion of a
stenotele, just below the level of the operculum, reveals the inter-
locking relationship of the stylets, previously described by Chap-
man and Tilney (ref. 4 Fig. 12). In the present figure, however, the
stylets are closer together and no spines are included in the section.

In an earlier paper (4), following the lead of previous workers
(e.g., ref. 7), it was stated that the capsular wall consists of
chitin or keratin. Since that paper was prepared, the author learned
of the work of Lenhoff ct al. (11), Johnson and Lenhoff (8) and
Lenlioff and Kline (10), in which it has been shown biochemical-
ly and histochemically that nematocyst capsules contain protein
which is probably a member of the collagen family of proteins.
Since one of the prominent fine structural features of collagen is
its characteristic periodicity, the electron micrographs of nemato-
cyst capsules were scrutinized carefully to determine whether any
indications of a periodic structure might be foimd. Figure 4, a trans-
verse section through the basal region of a stenotele, reveals the
presence, in the material of the invaginated capsular wall (ICW),
of a periodic structure with a 160 A periodicity. This is, of course,
a value one-fourth that of the usual 640 A period of collagen. It
should be noted that no fibrillar elements were observed in the
material of the capsule. Figure 5 shows a portion of the field of
Figure 4 at higher magnification. It is not immediately clear why
the invaginated capsular wall, which has been shown to be continu-
ous with the capsule proper (4), should reveal this periodic struc-
ture when the capsule proper does not. Nor is it clear why this

GEORGE B. CHAPMAN 135

periodic structure is so rarely observed. It may be, however, that the
preparative treatments usually fail to preserve this feature of the
structure. Figure 4 also includes a section through the enlarged
head of the tubule ( HD ) .

Figure 6 includes portions of two interstitial cells (I) and a
cnidoblast with a nearly mature stenotele. The cell at the lower left
is considered to be the least differentiated of the three cells, using
the criterion of sparse endoplasmic reticulum, as suggested by Slaut-
terback and Fawcett (12). That cell is of great interest, however,
because of the presence of a centriole ( CE ) with radiating spokes,
a configuration reminiscent of, yet somewhat different from, that
described in flagella by Gibbons and Grimstone (5). Although the
angle of section through the cnidoblast is inappropriate, the centri-
ole in the interstitial cell may be seen to bear a similarity to the
section through the base of the immature cnidocil ( CD ) . This
figure is, then, considered to indicate a relationship between the
centriole and cnidocil. Thus, the relationship between centrioles and
cilia and flagella may be extended to include cnidocils. ( More
extensive evidence in support of the differentiation of centrioles
into cnidocils will be presented elsewhere. However, it should be
noted here that Bouillon ct al. (2) believe the cnidocil to have a
structure anatomically distinct from that of cilia and flagella.)
Several elements of the outer supporting structures ( SP, ) and
inner supporting structures (SP^) of the cnidocil, as described by
Chapman and Tilney (3), are also visible in this figure.

Figure 7, an oblique section through the apical region of a
stenotele and a nearly transverse section through the cnidocil, also
reveals the similarity between the centriole and cnidocil base. Al-
though the state of preservation of this particular cnidocil renders
extensive discussion unwarranted, it may be stated that there ap-
pear to be nine peripheral groups of filaments, at least two of
which are composed of two elements, and a central group of three
filaments. In the original print, three circular profiles can just be
distinguished in the core of the cnidocil, once again reminiscent
of an appearance seen in flagella by Gibbons and Grimstone (5).
It should be noted that, even in their extensive study of flagellar
structure, Gibbons and Grimstone (5) apparently did not encount-

136 THE BIOLOGY OF HYDRA : 1961

er this central configuration. It may, then, constitute one more
variation from the basic pattern of filament arrangement in flagella
and cilia. The dense granules (G) resemble closely the granules
considered by Slautterback and Fawcett ( 12 ) to be glycogen.

SUMMARY

Electron microscopy of ultrathin sections of osmium-fixed and
Epon-embedded intact Hydra have revealed several new aspects
of stenotele fine structure. The internal tubule possesses an en-
larged head containing dense granules which may represent a por-
tion of the venom. The head has an extension ending in a hook.
The spines are seen to posses a bulbous enlargement at their
attachment to the invaginated capsular wall. Invaginated capsular
wall material showed a 160 A periodicity, possibly supporting the
belief that nematocyst capsules contain protein related to the
collagen family of proteins. A relationship is suggested between
centrioles and cnidocils (because of the similarity in appearance
of these two structures), thus extending the centriole— cilia, flagel-
la relationship.

REFERENCES

1. Bennett, H. S. 1960. Personal communication.

2. Bouillon, J., P. Castiaux, and G. Vandermeerssche. 1958. Structure submicro-

scopique des cnidocils. Bull. micr. appl., 8: 61.

3. Chapman, G. B., and L. G. Tilney. 1959. Cytological studies of the nemato-

cysts of Hydra. I. Desmonemes, isorhizas, cnidocils, and supporting struc-
tures. /. Biophysic. and Biochem. Cytol., 5: 69-78.

4. Chapman, G. B., and L. G. Tilney. 1959. Cytological studies of the nemato-

cysts of Hydra. II. The Stenoteles. /. Biophysic. and Biochem. Cytol., 5:
79-84.

5. Gibbons, I. R., and A. V. Grimstone. 1960. On flagellar structure in certain

flagellates. /. Biophysic. and Biochem. Cytol., 7: 697-716.

6. Hess, A. A., I. Cohen, and E. A. Robson. 1957. Observations on die structure

of hydra as seen with the electron and light miscroscopes. Quart. J. Micr.
Sc, 98: 315-326.

7. Hyman, L. H. 1940. The Invertebrates: Protozoa through Ctenophora, New

York, McGraw-Hill Book Company, 1-726.

8. Johnson, F. B., and H. M. Lenhoff. 1958. Histochemical study of purified

hydra nematocysts. ]. Histochem. and Cytochem., 6: 394.

GEORGE B. CHAPMAN 13:

9. Leeuwenhoek, a. 1702. In Antony van Leeuwenhoek and his "Little Animals,"
by Clifford Dobell, Dover Pubis. Inc., New York, 1960, p. 283.

10. Lenhoff, H. M., and E. S. Kline. 1958. The high imino acid content of the

capsule from Hydra nematocysts. Anat. Rec, 130: 425.

11. Lenhoff, H. M., E. S. Kline, and R. Hurley. 1957. A hydroxyproline-rich,

intracellular, collagen-like protein of Hydra nematocysts. Biochim. Bio-
phys. Acta, 26: 204-205.

12. Slautterback, D. L., and D. W. Fawcett. 1959. The development of tlie

cnidoblasts of Hydra. An electron miscroscope study of cell difterentiation.
/. Biophtjsic. and Biochem. Cytol., 5: 441-452.
1.3. Trembley, a. 1744. Memoires pour servir a Vhistoire d'lin genre de Polypes
d'eau douce, a bras en forme de cornes, Leyden, J. and H. Verbeck, 1-324.

14. Watson, M. L. 1958. Staining of tissue sections for electron microscopy with

heavy metals. /. Biophysic. and Biochem. Cytol, 4: 475-478.

15. Welsh, J. J., and M. Moorhead. 1960. The quantitative distribution of

5-hydroxy tryptamine in the invertebrates, especially in their nervous
systems. /. Ncurochcm., 6: 146-169.

Abbreviations used on Plates

capsule

mitochondrion

cnidoblast

nucleus

cnidocil

operculum

centriole

spine

granules, presumably

SPi

member of group of nine outer

glycogen

supporting structures

head of tubule

SP,

member of group of inner

hook of tubule

s'nporting structures

interstitial cell

stylet

ICW

invaginated capsular wall

tubule

matrix

In each figure, the mognification mark equols one micron.

Fig. 1. Sagittal section of a very nearly mature stenotele. Newly de-
scribed structures are the enlarged head of the tubule (HD), hook of the
tubule (HK) and the enlarged bulbous base of the spines (arrows). The
unique appearance of the material in the tubule head should be noted.
XI 7,000.

J 38

J 39

Fig. 2. Transverse section through the basal portion of a stenotele.
Bulbous enlargements of the bases of the spines are designated by arrows. A
portion of the enlarged head of the tubule (HD) is included in the section.
XI 5,600.

140

141

Fig. 3. Transverse section through the apical portion of a stenotele. The
interlocking stylet arrangement is shown. XI 7,000.

Fig. 4. Transverse section through the basal region of a stenotele. The
160 Angstrom periodicity of the invaginated capsular wall material may be
seen. The section also includes a portion of the head of the tubule. X29,000.

Fig. 5. An enlarged portion of Figure 4. X48,000.

142

143

Fig. 6. Section through two interstitial cells (I) and a cnidoblast (CB)
containing a nearly mature stenotele. A centriole (CE) may be seen in the
interstitial cell at the lower left. Note the centriole's radiating spokes. Mem-
bers of the outer (SP,) and inner (SP^.) groups of cnidocil (CD) supporting
structures may be seen. The cnidoblast cnidocil bears a resemblance to the
interstitial cell centriole. X17,000.

144

%r.

145

Fig. 7. Oblique section through the apical region of a stenotele and a
nearly transverse section through the cnidocil. The similar appearance
between this cnidocil base and the centriole of Figure 6 should be noted.
Granules, presumably glycogen, are designated G. X20,500.

146

. .\.

■^^^

147

148 THE BIOLOGY OF HYDRA : 1961

DISCUSSION

WOOD: Have you seen a periodicity in the internal supporting
structures which extend down past the capsule itself? I have seen
this several times, a periodicity reminiscent of the periodicity of
the ciliary rootlet found in other organisms but much narrower. I
was curious whether you had made a similar observation.

CHAPMAN: I have not seen any periodic structure there.

FAWCETT: For the benefit of those who are not electron
microscopists I would like to state that these pictures represent a
notable technical achievement. I think they show very clearly what
dramatic progress has been made in this field in a few years, largely
as a result of the introduction of new imbedding materials. A few
years ago, with all Dr. Chapman's skill, it was just impossible to
get such fine pictures of this very difficult object. Now with epoxy
resins, one can get beautiful micrographs of nematocysts and other
cytological features of hydra.

With respect to the localization of collagen or collagen-like
material— don't you feel that the very fine filaments found through-
out the substance of the nematocyst capsule may be tropocollagen
or collagen in such a state of dispersion that it would not be
expected to exhibit any periodic structure? The area which you
showed to have periodic cross banding may simply be a result of
side-to-side aggregation of such macromolecular units of collagen
that are more widespread than this limited occurrence of cross
striated structure would suggest.

CHAPMAN: This is very possible.

LENHOFF: The "collagen " of H. littoralis nematocysts is different
in many respects from vertebrate collagen. For example, there is
much more hydroxyproline and proline and also less glycine in the
nematocyst capsule than in classical collagens so that it is not sur-
prising that the periodicity is different. In fact, we are very
gratified that you find any periodicity at all.

WELSH: I wonder if this interesting structure that you show near
the tip of the external tubule might conceivably be a supporting
structure? I would seriously doubt that it was the toxin. Do you

GEORGE B. CHAPMAN 149

have any other evidence that the tips of the tubules attach to the
prey rather than penetrate?

CHAPMAN: I don't think there is any other evidence. There is
very Httle evidence on the structiue of the tubule itself, because
when tubules are fired they are hard as the dickens to find, and
when they are not fired we don't often get such sections as this one.

HAND: How many times have you seen that hook?

CHAPMAN: Just once. But we have other sections showing that
it is not an artifact.

HAND: You conclude that the tubule is not attached to the shaft
and basal portion of the extruded nematocyst?

CHAPMAN: No. That section did not show the hook hooked
on. It may never hook on. But it is suggestive. It's an interesting
arrangement. Why form a hook if you're not going to do something
with it?

MUSCATINE: Isn't it possible that the hook results from a
tangential section through a twist in the tubule?

CHAPMAN: If one examines the negative or a good print of
that particular figure very carefully, and specifically if one looks at
the membrane limiting that tubule, I think it would be conclud-
ed that the tubule had been cut very nearly sagittally and that it
probably is indeed a hook. It seems to have been fortuitously cut
precisely right. However, that is only an educated guess.

HAND: Which end of the thread is that really on?

CHAPMAN: It appears to be on the end that goes out first.

CROWELL: After the thread is fully discharged, which end is
the hook on?

CHAPMAN: It would be on the end which is farthest from the
stenotele.

HAND: Then you are proposing that that hook does not evert?

CHAPMAN: I am proposing that the tube which is coiled up
inside, which has a propeller-shaped cross section, may not evert.

150 THE BIOLOGY OF HYDRA : 1961

It may be fired out as a thread is fired out; as a fly line is fired out, if
you will.

HAND: Then it is not a homolog of the tube of the holotrichous
nematocyst such as Robson and Picken described?

CHAPMAN: I would agree.

HESS: I thought that it everted. Many previous investigators have
suggested this.

CHAPMAN: I can't possibly imagine a tube which is many
many microns long and is as narrow in diameter as that tul^ule is,
completely everting in the sense of eversion of the capsule itself. I
think it's almost impossible, even for a morphologist, to suggest any-
thing like that.

FAWCETT: I can, I have that kind of an imagination.

CHAPMAN : OK, you explain it.

FAWCETT: I am not going to explain the mechanism, but
direct your attention to the armament as it is folded within the
nematocyst. You'll agree that the armament has to turn inside out
in order to gain the position that it exhibits after the nematocyst
is fired. After firing, the part bearing the spines is smoothly continu-
ous with the wall of the tube and with the capsule. I think you'd
have a serious problem to explain it any other way. If you think
that only the base everts and the rest of it is flipped out like a fly-
rod, you would have to have a very different set of continuities
than you find in the fired nematocyst. I do not see how you can
have the base evert and the remainder flip out like a flyline. It has
to be one way or the other. A combination of the two mechanisms
is incomprehensible to me. I grant that it is difficult to visualize
how this entire tubule could turn inside out, but if the morphological
images suggest that this is true. I see no reason to doubt it simply
because there is no ready physical or chemical explanation. The
physical principals applicable to this problem may not have been
worked out yet.

CHAPMAN: Yes, but the problem involved in turning the en-
tire tubule inside out would be tremendous.

GEORGE B. CHAPMAN 151

CROWELL: I have thought for many years, hke Chapman, that
this turning inside out of so fine a tubule probably violated the rules
of hydraulics. It would be very nice to see a diagram of his inter-
pretation, perhaps compared with the conventional interpretation.

CHAPMAN: I may have to get a flyrod out of my car.

SLAUTTERBACK: We have often seen these stenoteles when
they have been interrupted by the fixative at various times during
the firing process. It can be seen clearly that the narrow part of
the tubule evaginates through the broad base, or butt, and expands
in diameter as it does so. Our observations would support the ele-
gant polarized light studies of Picken and Robson, whose interpre-
tations ought not to be neglected in this discussion. Furthermore,
since the base of the tubule is attached all the way around the
orifice of the capsule I cannot imagine a route of exit if you pro-
pose to get it out sideways.

WOOD: Nematocyst discharge is probably pretty much the same
for all nematocyst types. Using light microscopy, many people have
observed the tubes coming out from the inside of the discharging
holotrichs. To me, this is pretty strong evidence that the same
sequence occurs in the other nematocyst types. I would agree with
Dr. Slautterback.

CHAPMAN: My statements were made to explain how the tu-
bule might be discharged // the hook and swelling did in fact serve
the speculated functions. Most of the evidence supports the tubule
eversion hypothesis even though this eversion would involve a
nearly impossible physical feat.

Chemistry of Nematocyst

Capsule and Toxin

of Hydra Uttoralis

Edward S. Kline^' -

Biochemistry Branch, Arrived Forces Institute of Fathologij, Washington, D.C.

Nematocysts are highly organized, complex, intracellular struc-
tures. They are vital for the survival of the animal. Much of the
synthetic activity of the hydra is directed toward production of
the functional nematocyst, a structure that is quickly lost in the
course of its function in captiuing the live animals upon which the
hydra feeds. About 25% of the tentacle nematocysts are lost each
time H. Uttoralis eats a meal of Aiicmia; these are rapidly replaced
in about 48 hours from the store of differentiating nematocysts
in the body (11). Because of this continual and active develop-
mental process, nematocysts are an excellent system for chemical
studies of biosynthetic and morphogenetic processes. It is toward
an understanding of these processes that we hope to apply, as it
accumulates, information regarding the chemical structure of these
interesting organoids.

^ Studies of the author on the nematocyst capsule were carried out in collabora-
tion with Dr. H. M. Lenhoff, Howard Hughes Medical Institute, Miami, Florida;
studies on the succinoxidase inhibitor were carried out in collaboration with Dr.
V. S. Waravdekar, Chief of the Biochemistry Branch, Armed Forces Institute of
Pathology; studies on serotonin were carried out in collaboration with Dr. H. Weiss-
bach, Laboratory of Clinical Biochemistry, National Institute of Health, Bethesda,
Maryland.

2 Present address: Department of Chemistry, Indiana University, Bloomington,
Indiana.

153

154 THE BIOLOGY OF HYDRA : 1961

CAPSULE

Until recent years the nematocyst capsule was thought to be
chitinous (2) With the advent of the controlled mass culture
technique for the cultivation of H. littoraUs (13) it became feasible
to grow in the laboratory the large number of animals needed for
chemical studies on these structures. Under most conditions em-
ployed to disrupt Hydra one finds that most of the nematocysts dis-
charge. Advantage of this phenomenon was taken by Dr. LenhofI,
who, by a procedure of differential centrifugation, was able to iso-
late nematocyst preparations, largely discharged and free of the bulk
of other tissue components (Table 1). When we chromatographed

TABLE 1
Isolation of discharged nematocysts from Hydra

1. Live animals disrupted in 10 kc sonic oscillator.

2. Suspension centrifuged 15 minutes at 160 g. Supernatant recentrifuged.

3. Whitish residues pooled and resuspended in water.

4. Residues centrifuged 20 minutes at 200 g.

5. Residue contained at least three types of nematocysts— penetrants, volvents, and

glutinants — about 80% are discharged.

hydrolysates of such a preparation we found numerous amino acids,
among them, one which was identified chromatographically as
hydroxyproline (9). This compound, to my knowledge, has never
been demonstrated in any protein other than collagen. The quanti-
tative distribution of this compoimd between the protein of the
whole Hydra and the nematocyst preparation shows that it is con-
centrated in the latter ( Table 2 ) . We have also found high amounts
(over 20%) of the other imino acid, proline, in the nematocyst prepa-
rations (10). The absolute values obtained for the imino acid con-
centration in nematocyst preparations have shown some variation.
More recent determinations of the hydroxyproline indicate that it
is present in even higher concentrations than shown in the above
table. These differences can be largely due to variations in the
purity of different preparations. Since, in animals, high concen-
trations of these imino acids have been demonstrated only in
collagens, we conclude that the capsule of the Hydra nematocyst

EDWARD S. KLINE 155

TABLE 2
Hydroxyproline contend of whole Hydra and of nematocyst protein (From ref. 9)

Fraction Hydroxyproline Nitrogen g. Hydroxyproline

(mg. ) (mg. ) per 16 g. N

Hot — TCA — precipitate

from 6,800 hydranths 3.568 5.21 1.74

Nematocyst preparation 0.230 0.285 12.7

contains a protein belonging to this class of componnds. The
isolated nematocysts exhibit JDirefringence, visually resist trypsin
digestion, show extreme stability to autoclaving, stain blue with
the Masson trichrome reagent, show metachromasia with toluidine
blue, and stain positively with alcian blue (9, 3). Except for the
heat stability exhibited by the nematocyst preparation, all of these
properties are indicative of a collagen type structure. One of the
best known characteristics of the classical collagens is its property
of forming gelatin upon heating, yet the nematocyst capsule retains
its morphology even after many hours in the autoclave at 121'.
I do not know whether this is due to the presence of other material
in the capsule that "holds it together" or to the intrinsic inertness
of the hydroxyproline-containing protein. Other lines of evidence
have led Phillips ( 16 ) to postulate that the capsule of the anemone,
Metridiiim senile, contains a cartilaginous material.

TOXIN

In connection with the work carried forth in our laboratory,
the word "toxin" is probably inappropriate. I use it, only for the
lack of a better term, to denote any nonstructural material which
we believe to be present inside the nematocyst. We have no definite
information tha^ these materials constitute a toxin or even part
of a toxin.

Stwcinoxidase Inhibitor — In an attempt to correlate the activity
of oxidative enzymes with the phenomenon of regeneration we
found that succinoxidase activity is virtually absent in Hydra homo-

156 THE BIOLOGY OF HYDRA : 1961

genates (Table 3). This observation led to the realization that
Hydra extracts contain an inhibitor of this enzyme system (6). It
seemed reasonable to look for the inhibitor in the nematocysts,
since these structures could contain this substance and thus prevent
it from affecting the succinoxidase of the intact Hydra.

TABLE 3
Demonstration of succinoxidase inhibitor in Hydra littoralis (from ref. 6)

Tissue homogenate Succinoxidase activity

Qoo (dry weight)"

Hydra

Mouse liver

Q02 ( wet weight ) *

Mouse liver

Mouse liver plus extract from Hydra*

2.4

^Qo2 = /A of O2 consumed per hour per mg. of tissue.

*The extract was obtained from Hydra suspended in distilled water. The animals

were disrupted in a 10 kc Raytheon sonic oscillator, and most of the nematocysts

and nematocyst walls were removed by centrifugation.

In all reaction vessels 0.1 ml. of a 5% homogenate of liver was used.

A basic difficulty that has characterized nearly all of the studies
connected with coelenterate toxins has been the unavailabihty of
isolated, undischarged nematocysts. Most studies have been carried
out on toxic material obtained either from the whole animal or from
tentacles and acontia. At the time our study was performed we
also were unable to isolate clean, undischarged nematocysts. There-
fore, our evidence showing that we are dealing with nematocyst
material is indirect. Rather than to compare the amounts of in-
hibitory material in the various parts of Hydra, we attempted
to elicit nematocyst discharge from the live, intact animal and see
whether inhibitory activity is present in the culture Huid surrounding
the animals. Chemical compounds known to cause nematocyst
discharge were ineffective in our hands. Generally, the concentra-
tions needed to cause the response also caused great damage to the
animals. We did find, however, that a shock from a dry cell battery
would induce discharge without killing the animals and with con-

EDWARD S. KLINE 157

siderably less \isual tissue damage than is caused by the chemical
agents. As a result of the electric shock, significant numbers of
nematocysts are discharged and soluble material is released into
the culture medium; material which inhibits succinoxidase of mouse
liver (Table 4). The possibility exists that this procedure causes
the release of soluble material other than that from nematocysts.
A. similar way of causing nematocyst discharge has been reported
by Glaser and Sparrow ( ] ) . These workers also jDresented a pro-
cedure for the isolation of undischarged nematocysts from hydra
and other coelenterates. More recently Phillips (16, 17) and Lane
and Dodge (8) have presented procedures for the isolation of
undischarged nematocysts from Metridium and Physaha, respec-
tively. By modifications of their methods we are now able to collect
undischarged nematocysts from Hydra littoralis and hope to study
these soon.

We purified the inhibitor ( Table 5 ) and attempted to learn some-
thing of its nature. The purified inhibitor is a macromolecule
which appears as a slow-moving and apparently rapidly diffusing
single peak in the ultracentrif uge ( Fig. 1 ) . Ultracentrifuge experi-

TABLE 4

Succinoxidase inhibition by material discharged from nematocysts by
electric current (from ref. 6)

Inhibition of
Material enzyme activity

Mouse liver homogenate plus Hydra medium

before electric shock (0.4 ml.) 2

Mouse liver homogenate plus Hydra medium

after electric shock ( 0.4 ml. ) 27

There were 57 animals in 2.5 ml. of solution. Aliquots of the solution without
Hydra were removed before the shock. The solution was then diluted to the original
volume and a shock from a 45-volt dry cell battery was applied. Then another aliquot
of the solution was removed. For the succinoxidase assay of 0.1 ml. of a 5% liver
homogenate was used in tlie reaction vessel. Q02 ( wet weight ) of control liver = 25.
The same overall effect can be repeated but the actual precentage of inhibition does
vary, depending upon differences in experimental conditions, e.g., the time the shock
is applied and the number of nematocysts that are discharged.

158

THE BIOLOGY OF HYDRA : 1961

Fig. 1. Ultracentrifuge pattern of purified inhibitor at 59,000 r.p.m. (6).

ments indicate the molecular weight to be less than 50,000.
Chemical tests show the presence of protein. The ultraviolet
absorption spectrum (Fig. 2) is qualitatively very similar to that
produced by proteins such as serum globulins. There are no
peaks in the visible spectrum. All of our characterization studies
affirm the presence of a protein in the purified inhibitor and, as

0.8 ~r

240 250 260 270 280 290 300 310 320 330 360

WAVE LENGTH (tu^)

Fig. 2. The ultraviolet absorption spectrum of the purified inhibitor in
phosphate buffer (6).

EDWARD S. KLINE

159

TABLE 5
Fractional separation of succinoxidase inhibitor from Hydra (from ref. 6)

Lyophylized homogenate

Extracted with n-butvl alcohol

Soluble (inactive)

Residue ( active )
Extracted with acetone

Residue (active)
Extracted with ether

Soluble (inactive) Residue (active)

Extracted with 0.02 M phosphate buffer, pH 8

Phosphate soluble

( about 92% of the activity ) '

pH adjusted to 3.9

Residue

( about 8% of the activity )

Supernatant

Dialized against 0.02 M phosphate buffer, pH 8

Fraction A
( about 75% of activity in "phosphate soluble" fraction )
Centrifuged in Spinco preparative ultracentrifuge

Residue— discarded

Supernatant— purified inhibitor
(Almost all of activity in Fraction A)

'Sample dialyzed before activity determination.

Residue— discarded

J 60 THE BIOLOGY OF HYDRA : 1961

yet, we have not detected any other class of compounds. Next, we
thought it of interest to determine whether the succinoxidase in-
hibitory activity is associated with the integrity of the protein. This
was studied by incubating the purified inhibitor with the proteolytic
enzyme trypsin to see whether digestion by this enzyme reduced
the inhibitor's activity. After the incubation, soybean trypsin inhib-
itor was added to destroy the trypsin activity. The purified inhibitor,
after such treatment, lost virtually all of its activity against mouse
liver succinoxidase (6). From these studies we concluded that the
inhibitor is a protein and that a high degree of the integrity of the
protein is required for its activity.

At this stage of the study we became interested in the nature
and mechanism of the succinoxidase inhibition produced by the
Hydra protein. The next few experiments describe some of our find-
ings in this area (7). The main parts of the succinoxidase chain,
as it is now believed to exist, is shown in Figure 3. It is possible

SUCCINATE ->► SUCCINIC DEHYDROGENASE-

-^CYT. c, ->► CYT. c >

Fig. 3. A pathway for the oxidation of succinate. Not shown are the
fat-soluble factors implicated in succinate oxidation, e.g., coenzyme Q,
tocopherol and vitamin K.

by the use of specific assays to locate in this chain the general and
perhaps the exact site at which an inhibitor acts. The quantitative
effect of the purified inhibitor on succinoxidase is presented in
Figure 4. These data show that the inhibition is linear to about the
50% level. The maximum level of inhibition is less than 100%,
although, in other experiments the 100% level has been reached. The
primary portion of the succinoxidase system, succinic dehydro-
genase, does not appear to be the area in which the Hydra inhibi-
tor operates ( Table 6 ) . Here, with 48 times the amount of material
needed to inhibit succinoxidase 50%, there is less than 20% inhibition.
The terminal portion of the succinoxidase chain, cytochrome oxi-
dase, is not inhibited at all by 22 times the 50% inhibitory level for
succinoxidase (Table 7). The next subsystem that we studied was
succinate-cytochrome-c reductase. This system probably includes a

EDWARD S. KLINE

161

.04

.08

.12 .16 .20 .24 .28

ML. OF PURIFIED INHIBITOR

.36

Fig. 4. The inhibitory effect of the purified inhibitor on the succinoxidose
activity of mouse liver homogenate. In both Curve A and Curve B, the flasks
contain 0.1 ml. of liver homogenate (5 mg. of wet tissue per flask) and
the purified inhibitor (2.9 /;g. of protein nitrogen per ml. of inhibitor). Total
volume in all flasks: 3.1 ml. Each curve represents separate experiments (6).

TABLE 6
Effect of the purified inhibitor on succinic dedydrogenase

Material

Inhibitor to Succinic dehydrogenase Inhibition
tissue ratio activity

Hg.: mg. Q02 (wet weight) %

Mouse liver homogenate
Mouse liver homogenate
plus purified inhibitor
( 100 ^g. protein N )

2.5 : 1

4.0

3.3

17.5

Each vessel contained 40 mg. of aqueous liver homogenate in final volume
of 2.9 ml.

162 THE BIOLOGY OF HYDRA : 1961

TABLE 7
Action of the purified inhibitor on cytochrome oxidase

Material Inhibitor to Cytochrome Inhibition

tissue ratio oxidase activity

fig.: nig. Qoo (wet weight) %

Mouse Hver homogenate ■ 96

Mouse liver homogenate

plus purified inhibitor

(1.16 /ig. protein N) 1.16 : I 110 0.0

Each vessel contained 1.0 mg. of aqueous liver homogenate in final volume of
2.9 ml.

flavin moiety and components of the intermediary portions of
succinoxidase, terminating at cytochrome c. The exact nature of
this system in the enzyme preparation we used is not thoroughly
understood. If the purified inhibitor is preincubated with the mouse
hver homogenate, we find significant inhibition of succinate-cyto-
chrome-c reductase. The inhibition, although less than with succin-
oxidase, is pronounced and occurs with the concentration of inhib-
itor which produces 50% reduction of succinoxidase (Table 8).

Our overall results in this area of the investigation lead us to
postulate that the inhibition is specific [based on criteria of Keilin
and Hartee ( 4 ) and Slater ( 22 ) ] and that the reduction of cyto-
chrome c is blocked, that is, the inhibition occurs on the substrate
side of cytochrome c. Fmthermore, since there is no evidence for

TABLE 8
Effect of the purified inhibitor on succinate-cytochrome-c reductase

Material Inhibitor to Reductase activity Inhibition

tissue ratio ( cyt. c reduction at 550 m/x )
fig.: mg. fi moles in 10 min. %

Mouse liver homogenate 0.0237 ■

Mouse liver homogenate

plus purified inhibitor

(0.0116 /ig. protein N) 0.058 : 1 0.0209 12

Mouse liver homogenate

plus purified inliibitor

( 0.0309 /ig. protein N) 0.174 : 1 0.0094 60

Aqueous liver homogenate preincubated with purified inhibitor for 30 minutes
at room temperature. Each cuvette in assay had 0.2 mg. of homogenate. Volume in
each cuvette = 3.0 ml.

EDWARD S. KLINE 163

direct inhibition of cytochrome b or any component preceding cyto-
chrome b, we postulate that the inhibition occurs between cyto-
chrome b and cytochrome c, ( based on scheme in Fig. 3 ) . This part
of the system is not thoroughly characterized and I will not attempt
to expand further on the above conclusions, except to add that these
conclusions are similar in many ways to those drawn by Slater in
his work with BAL (21, 22, 23), by Potter and Reif with antimycin
A (18, 19, 20), and by Lightbown and Jackson with 2-heptyl-4-
hydroxy-quinoline N oxide ( 12 ) .

The purified inhibitor from Hydra has toxic effects on the
mouse and the fiddler crab (5). When injected with the inhibitor
(10 micrograms per gram body weight) most or all of the fiddler
crabs became sluggish and about one-half of them lost their ability
to right themselves, when placed on their backs. Eventually some
of the animals died but most recovered. These effects were opposed
to those of the boiled inhibitor, with which little or no adverse
affects were noticed. We have done only a small number of experi-
ments of this kind, with only 5 to 6 animals in each group, thus,
we cannot really say much about the toxicity except that it occurs.
Based on the amount of inhibitor required to elicit a discernible
response in both the mouse and crab it does not appear that this
material can account for more than a portion of the potent effect
of toxic material present in Hydra. Welsh and Frock (25) have
found tetramethylammonium in Hydra littoralis. If this compound
is present in the nematocysts I expect that it may account for a large
measure of the toxins potency.

Hydroxijindoleamines — Both hydroxyindoles and hydroxyin-
doleamines have been demonstrated in various coelenterates ( 14-17,
24, 26). One of these reports contained studies on H. oligactis (26).
In it, Welsh showed the presence of significant amounts of
5-hydroxytryptamine (serotonin) in homogenates of this animal.
Dr. Weissbach and I have found high concentrations of a 5-hy-
droxytryptamine in Hydra littoralis. We induced nematocyst dis-
charge by electric shock and compared the amounts of 5-hydroxyin-
doleamine in the Hydra medium with that present in the whole
animal (Table 9). This experiment showed that the discharged
hydroxyindoleamine was present in more than 10 times the con-
centration than was found in the whole animals. Several attempts

164 THE BIOLOGY OF HYDRA : 1961

TABLE 9
Hydroxyindoleamine distribution in fractions from Hydra littoralis

Preparation Hydroxyindoleamine concentration

fig. per ml. of preparation ^g. per gram dry

tissue

Whole Hydra homogenate 2.56 52

Hi/dra culture medium— before

shock 0.011 57

Hydra culture medium— after

shock 0.27 534

Conditions for the shock experiment similar to those used for experiment shown
in Table 4.

were made to determine if the amine was serotonin. We have dem-
onstrated a 5-hydroxyindoleamine by paper chromotography but
as yet have not been able to obtain a sufficiently clean extract
to determine precisely the identity of the compound. We do have
indirect evidence that the compound is serotonin rather than bufo-
tenine. This evidence consists of partition coefficients between ether
and an alkaline aqueous phase. A 5-hydroxytryptophan decar-
boxylase is present in Hydra but this only shows that the animal can
synthesize serotonin, not that serotonin is there. We are inclined to
believe that serotonin is present in Hydra littoralis, but direct proof
is still lacking.

CONCLUDING STATEMENTS

This study represents but a start toward the elucidation of the
chemical composition of the nematocyst. It will be of interest not
only to further characterize such preparations as the ones we
have studied, but also to separate and compare the various types
of nematocysts present in hydra, as well as the components of each
nematocyst type. This information, coupled with the excellent
morphology studies that are being carried out in various labora-
tories, could form the basis for an understanding of the manner in
which these intriguing organoids develop and function.

EDWARD S. KLINE 165

REFERENCES

1. Glaser, O. C, and C. M. Sparrow. 1909. The physiology of neniatocvsts. /.

Exp. Zool. 6: 361-382.

2. Hyman, L. H. 1940. The Invertehraies: Protozoa through Ctcnophora. McGraw-

Hill Book Co., Inc., New York, p. 382.

3. Johnson, F. B., and H. M. Lenhoff. 1958. Histochemical study of purified

Hydra nematocysts. /. Hist. Cytochem. 6: 394.

4. Keilin, D., and E. F. Hartree. 1949. Activity of the succinic dehydrogenase —

cytochrome system in different tissue preparations. Biochem. J. 44: 205-218.

5. Kline, E. S., and V. S. Waravdekar. 1959. Toxic effects of a material isolated

from Hydra littoralis. Amer. Soc. Pharmacol. Exp. Therap. 1: 62.

6. Kline, E. S., and V. S. Waravdekar. 1960. Inhibitor of succinoxidase activitv

from Htjdra littoralis: J. Biol. Chem. 235: 1803-1808.

7. Kline, E. S., and V. S. Waravdekar. 1960a. On the site of action of a suc-

cinoxidase inhibitor from Hydra. Fed. Proc. 19: .35.

8. Lane, C. E., and E. Dodge. 1958. The toxicity of Physalia nematocysts. Biol.

Bull. 115: 219-226.

9. Lenhoff, H. M., E. S. Kline, and R. Hurley. 1957. A hydroxyproline-rich,

intracellular, collagen-like protein of Hydra nematocysts. Biochem. Biophys.
Acta. 26: 204-205.

10. Lenhoff, H. M., and E. S. Kline. 1958. The high imino acid content of the

capsule from Hydra nematocysts. Anat. Rec. 130: 425.

11. Lenhoff, H. M., and J Bovaird. 1961. A quantitative chemical approach to

problems of nematocvst distribution and replacement in Hydra. Develop.
Biol. 3: 227-240.

12. Lightbown, J. W., and F. L. Jackson. 1956. Inhibition of cytochrome systems

of heart muscle and certain bacteria by the antagonists of diliydrostrepto-
mycin: 2-alkyl-4-hydroxyquinoline N-oxides. Biochem. J. 63: 130-137.

13. LooMis, W. F., and H. M. Lenhoff. 1956. Growth and sexual differentiation

of hydra in mass culture. /. Exp. Zool. 132: 555-568.

14. Mathxas, a. p., D. M. Ross, and M. Schachter. 1957. Identification and dis-

tribution of 5-hydroxytryptamine in a sea anemone. Nature 180: 658-659.

15. Mathias, a. p., D. M. Ross, and M. Schachter. 1960. The distribution of 5-

hydroxytryptamine, tetramethylammonium, homarine, and other substances
in sea anemones. /. Physiol. 151: 296-311.

16. Phillips, J. H. 1956. Isolation of active nematocysts of Metridium senile and

their chemical composition. Nature 178: 932.

17. Phillips, J. H., and D. P. Abbott. 1957. Isolation and assay of die nematocyst

toxin of Metridium senile fimhriatum. Biol. Bull. 113: 296-301.

18. Potter, van R., and A. F. Reif. 1952. Inhibition of an electron transport com-

ponent by antimycin A. /. Biol. Chem. 194: 287-297.

19. Reif, A. F., and van R. Potter. 1953. Studies on succinoxidase inhibition:

1. Pseudoreversible inhibition by a napthoquinone and by antimycin A.
;. Biol. Chem. 205: 279-290.

20. Reif, A. F., and van R. Potter. 1954. Oxidative pathways insensitive to anti-

mycin A. Arch. Biochem. 48: 1-6.

21. Slater, E. C. 1948. A factor in heart muscle required for the reduction of

cytochrome c by cytochrome h. Nature 161: 405-406.

22. Slater, E. C. 1949. The action of inhibitors on the system of enzymes which

catalyze the aerobic oxidation of succinate. Biochem. J. 45: 8-13.

166 THE BIOLOGY OF HYDRA : 1961

23. Slater, E. C. 1949. A respiratory catalyst required for the reduction of cyto-

chrome c by cytochrome b. Biochem. }. 45: 14-30.

24. Welsh, J. H. 1955. On the nature and action of coelenterate toxins. Deep Sea

Research, Suppl. 3: 287-297.

25. Welsh, J. H., and P. B. Prock. 1958. Quaternary ammonium bases in the coelen-

terates. Biol. Bull. 115: 551-561.

26. Welsh, J. H. 1960. 5-Hydro.xytrytamine in coelenterates. Nature 186: 811-812.

DISCUSSION

WELSH: Were these serotonin values on a dry weight or wet
weight basis?

KLINE: Dry weight basis.

WELSH: What do you say about the heat and pH stabihty of the
succinoxidase inhibitor?

KLINE: It is stable at pH 5.8 and 8, and since one step in the
purification of the inhibitor is a pH 4 precipitation it has appre-
ciable stability even at this pH.

Heat stability is an interesting point. We felt that the inhibitor
from Hydra littoralis could have been a phospholipase A. Phospho-
lipase As are heat stable and the succinoxidase inhibitor in snake
venom is believed to be this enzyme. We heated separately some
Crotalus adamanteus venom and our inhibitor at pH 5.8 in a boil-
ing water bath for 15 minutes. The venom lost none of its effec-
tiveness against succinoxidase while the purified inhibitor from
Hydra lost about 75% of its activity.

EAKIN: What is its behavior on dialysis?

KLINE: Essentially all of the activity is non-dialyzable.

LENHOFF: Does the inhibitor do anything to mitochondria?

KLINE: We have done one or two preliminary studies and there
seems to be some effect on the mitochondria, but as yet we have
done too little to make any definite statements.

MARTIN: Have you ever tried to extract active substances from
the nematocyst-poor parts of the Hydra? And if so, did they show
any similarity with the nematocyst content?

EDWARD S. KLINE 167

KLINE: Which component? Serotonin?

MARTIN: Serotonin or the enzyme inhibitor.

KLINE: We have not done that. I beheve that the best proof for
the locahzation of these compounds will come when we can
quantitatively isolate pure, undischarged nematocysts from the
animal.

ROSS: I'm very interested and pleased to see your results with
serotonin. But I'd like to hear your comments on some observations
that Mathias, Schachter, and I made in London on the distribution
of serotonin in sea anemones, because our results would indicate
that we cannot extend this conception generally over a whole group
from one species unless one looks at the distribution very care-
fully. We found extracts from tentacles separated from the column,
or both separated from the tissues lining the coelenteron did not
contain much serotonin in 3 of the 4 species of sea anemones
that we used, viz., Metridiiim senile, Actinia equina and Anemonia
sulcata. The only place where we found a significant amount of
serotonin was in the "coelenteric tissue" of CalUactis parasitica,
and there it was present in large quantities, 500-600 mg. per gram
of freeze-dried matter. This was about 60 times the concentration
found in the tentacles. Thus there seemed to be no correlation
between the distribution of serotonin and nematocysts, or be-
tween different species. I wonder if you have any comment to
make on that?

KLINE: I am aware of your work and it might appear that the
findings in various laboratories are contradictory. But as you have
said, we cannot necessarily extend results from one animal to an-
other. I believe Phillips thought his hydroxyindoleamine was
bufotenin rather than serotonin and that it was not localized in the
nematocysts. Your group found serotonin in certain anemones and
not in others and you feel that it is not concentrated in the nemato-
cysts. Is this correct?

ROSS: Well, it's in a part of the animal where there are fewer
nematocysts.

KLINE: Dr. Welsh's study with anemones points to it being
serotonin and in the nematocysts. For the most part we all have

163 THE BIOLOGY OF HYDRA : 1961

been studying different animals with different approaches. As time
passes I become more impressed by the variabiHty between closely
related animals.

PHILLIPS: Have you detected any hexosamines or uronic acids
in the capsule?

KLINE: We have not looked for them.

STREHLER: What percentage of the total weight did you calcu-
late would be collagen on the basis of this hydroxyproline
content?

KLINE: Based on 20% hydroxyproline, the collagen-like protein
represents about 10% of the total protein of H. littoralis.

Physalia Nematocysts and
their Toxin

Charles E. Lane

Institute of Marine Sciences, University of Miami, Miami, Florida

Nematocysts in Physalia are widely distributed through the
epithehum clothing most of the members of the colony. These
organelles are formed in cnidoblasts by so far imdescribed cyto-
genetic processes. The upper surface of the float and the proximal
portions of the gastrozooids and of the fishing tentacles are relatively
deficient in mature nematocysts. Over the surface of the fishing
tentacle cnidoblasts are concentrated in the epithelium clothing
the batteries. These are permanent structures distributed in bead-
like fashion along the length of one edge of the tentacle, and they
are illustrated in Figure 1, which shows a three-dimensional re-
construction of a segment of the fishing tentacle of Physalia. The
batteries appear as discrete saccular enlargements along one edge.
A longitudinal section through a portion of the tentacle, including
a single battery cut equatorially appears in Figure 2.

The battery is lined by gastrodermis continuous with that
lining the gastrovascular extension in the tentacle. The mesoglea
is a thick band of fibrous connective tissue external to the gastro-
dermis. The epidermal layer bearing cnidoblasts clothes the entire
structure. At the equator of the battery the epidermis thickens
abruptly where the external hemisphere acquires its population of
mature cnidoblasts. Perhaps the most outstanding histological char-
acteristic of this epithelium is the regular distribution through
it of nematocysts belonging to two different size groups. The total
thickness of the epithelium is just sufficient to clothe the large
nematocysts, which range from 25 to 30 microns in diameter.

169

170

THE BIOLOGY OF HYDRA : 1961

Fig. 1. Reconstruction in wax of a segment of the fishing tentacle of
Physalia. The extension of the gastro-vascular cavity into the tentacle and
the relationship between the cavities of the batteries and of the tentacle are
clearly shown.

Regularly spaced between cnidoblasts bearing the large nemato-
cysts occur small cnidoblasts whose capsules range from 7 to 15
microns in diameter. In favorable preparations each of the cni-
doblasts may be seen to be provided with a cnidocil, projecting
through the cuticular layer of the epithelium into the ambient water.
The light microscope reveals a perinuclear basketwork of elastic
fibers within the cnidoblast, which appears to surround the nema-
tocyst. Other than this perinuclear network and the nematocyst
capsule, the cytoplasm of the definitive cnidoblast appears to pre-
sent very little structural specialization.

Cnidoblasts are regularly distributed throughout the epidermis
of the external battery hemisphere, and there is also a repeating
pattern of internal structure in adjacent nematocysts. The internal
coiled thread, characteristic of the nematocysts of all Cnidaria, in
each of the nematocysts originates at about the same point in the

CHARLES E. LANE

171

l4»

Fig. 2. Frontal Section through the fishing tentacle of Physalia X 200.
The gastrovascular cavity of the tentacle communicates in the center of the
field with the cavity of a single "battery." Hypertrophy of the gastrodermis
begins at the equator of the battery.

capsule and coils in the same clock-wise direction in approximately
the same plane.

If the surviving tentacle be stimulated by gradually increasing
the concentration of solutes in the surrounding water, the nema-
tocysts may be made to discharge. This is a dramatic, explosive
process, the nematocyst threads being hurled from the capsule
with sufficient force to penetrate the surface film. This observation
explains our early experience of being severely stung even through
a surgical glove.

In our laboratory we isolate surviving nematocysts by con-
trolled autolysis at 4", followed by screening, sieving, washing,
and settling. The washing process is continued until the wash
water is no longer toxic when injected into the hemocoele of the
fiddler crab. This point may acquire some significance when one
attempts to compare the activity and biochemistry of the toxin

172

THE BIOLOGY OF HYDRA : 1961

prepared in our laboratory with reports in the hterature describing
the activity of other Physalia toxin preparations. Earher investiga-
tors, ahnost uniformly, have homogenized and extracted entire
tentacle material. It will later appear that there are active extra-
nematocyst substances present in the tentacle; however, the bio-
chemistry and pharmacology of these materials has not been
studied in our laboratory.

Isolated nematocysts may be concentrated by settling and de-
cantation of the supernatant water. They do not survive centri-
fugation without discharge so it is necessary to permit them to
settle by gravity alone. The putty-like concentrate resulting from
our procedure is virtually free of tissue fragments and contains
very few (less than 1%) discharged nematocysts. The concen-
trated nematocysts are frozen and stored in the deepfreeze where
they retain their reactivity for periods of at least four years.

Surviving nematocysts are homogenized in an all-glass homog-
enizer in a minimum volume of distilled water for about twenty
minutes, or until an aliquot shows no more than 10% unbroken
capsules. The resulting brei is centrifuged at 4° in a refrigerated
centrifuge at 15,000 > gravity; the residue is resuspended in a

TABLE 1
Amino acids in an acid hydrolysate of the crude toxin

Amino

^xA//Sample

Relative

Acid

Concentration

Alanine

0.37

Arginine

0.12

Aspartic Acid

0.32

Glutamic Acid

0.85

Glycine

0.75

Histidine

0.05

Isoleucine

0.19

Leucine

0.25

Lysine

0.25

Phenylalanine

0.15

Proline

0.38

Serine

0.23

Threonine

0.07

Tyrosine

Valine

0.21

CHARLES E. LANE 173

minimum of water and recentrifuged. The supernatant solutions
from these two centrifugations are combined and lyophiUzed.

The lyophihzed "crude" toxin has regularly assayed between
15 and 16'i nitrogen by micro-Kjeldahl. All tests for polysaccharide
have been negative. A sample of crude toxin was hydrolyzed in
6N HCl, and the hydrolysate was analyzed on the Beckman Spinco
amino acid analyzer with the results shown in Table I.

The lyophihzed toxin is lethal to mice at dosage levels of 1.7
mg. kilogram.

When crude toxin was chromatographed one-dimensionally with
80^ »-propanol as the solvent system, a series of nine spots ap-
peared when the paper was developed with ninhydrin. Each of
the spots was separately eluted and assayed for total activity in
the fiddler crab, Uco piigilafor. Four of the spots accounted for
95% of the total biological activity of the crude toxin.

The active regions on the chromatographic papers were eluted,
hydrolyzed, and rechromatographed. Each was shown to contain
more than one amino acid.

Since this chromatography had been accomplished in the pre-
sence of a solvent and at room temperature it was felt that consid-
erable loss of activity may have occurred. Such an attenuation
might be sufficient to mask activity in other fractions. Accordingly,
the crude toxin was next fractionated on the Beckman refrigerated
paper curtain electrophoresis apparatus, using phthallate buffer pH
5.8 at 2". Four fractions were separated; after dialysis and lyophili-
zation they were carefully diluted to their relative concentration
in the original toxin and bioassayed on Uca pugilator. The
results are shown in Figure 3.

One peptide nearly equals the activity of the original whole
toxin, although representing less than 10% of its weight, and it
therefore appears that some inert masking protein materials may
have been removed by electrophoresis. Physalio toxin, therefore,
appears to he a relatively simple protein consisting of only a few
toxic peptides. Our future studies will seek to describe the precise
molecular configuration of these peptides and to relate biochemical
structure to pharmacologic activity.

I may be permitted to speculate briefly about the origin and
synthesis of Physalia toxin. The gastrodermis lining the battery

174

THE BIOLOGY OF HYDRA : 1961

Toxin Fraction

Fig. 3. Results of bioassay on groups of 10 ilea pugilator of: (0) whole
crude toxin, and various fractions (1, 2, 3, 4) separated by electrophoresis
from the crude toxin. Fractions were injected at levels approximating their
separate concentration (by weight) in the crude toxin.

undergoes characteristic hyperplasia beneath that portion of the
epidermis containing mature nematocysts. This histological change
involves structural polarization, extensive vacuolation of the cyto-
plasm, and a change in the staining characteristics and chromatic
density of the nucleus (see Fig. 2). The mesoglea separating the
hyperplastic gastroderm cells from the nematocyst-containing epi-
dermis is also modified. In preparations stained with Mallory's
trichrome, the mesoglea shows discrete circular patches which stain
differently from the rest of the mesoglea. These patches are always
located between hypertrophied gastroderm cells and cnidoblasts
in the surface epithelium.

We have shown the gastrovascular cavity of Physalia to contain
and circulate a protein fluid. There is open communication between
the gastrovascular cavity and tlie cavity of the battery. It is tempting
to speculate that the modified gastrodenn cells basal to the cnido-

CHARLES E. LANE 175

blasts absorb precursor materials from the circulating gastrovascular
fluid and from these synthesize the toxin which they subsequently
secrete through the mesoglea and into the cnidoblasts.

Slautterback and Fawcett have shown that the nematocyst thread
in hydra originates from outside the nematocyst capsule and is
subsequently introduced into the cavity of the nematocyst. If a
structural component of the nematocyst may be formed external
to the nematocyst capsule and subsequently introduced into it, it
should not stretch our credulity too far to accept the suggestion
that a soluble protein toxin may be synthesized outside the nema-
tocyst and later may pass through it. One disturbing observation
is that this would suggest or almost require that the nematocyst
capsule be permeable to the toxin. We have repeatedly observed
that the toxin does not leach from surviving purified nematocysts.
Presumably, therefore, there is one stage in the morphogenetic
history of the nematocyst when the capsule wall may be permeable
to toxin but in the mature nematocyst these permeability relation-
ships may be completely changed.

I suggest that Phijsalia toxin is synthesized by gastrodermal
cells, passes through the mesoglea, and into the nematocyst during
the morphogenesis of this structure.

DISCUSSION

CROWELL: Where are the nematocysts manufactured in Phijsa-
lia? Where is the differentiation of the cnidoblasts taking place?

LANE: I can't answer because I don't know yet. I can tell you
a few of the things we do know. In adult animals the float is
generally free of cnidoblasts. The basal ends of gastrozoids are
deficient in cnidoblasts. Cnidoblasts appear to be reasonably uni-
formly distributed throughout the length of the fishing tentacle. I
have seen no clear histological evidence of interstitial cells such
as we have heard about in hydra. Obviously they must be there,
but I haven't seen them.

CROWELL: Is it possible that the whole tentacle is continuously
growing so that it's always young basally and degenerating api-

176 THE BIOLOGY OF HYDRA : 1961

cally? If so, there is no need to replace the nematocysts along
the length of the tentacle.

LANE: I think this is entirely possible.

CROW ELL : Two other possibilities are that nematocysts are made
all along the tentacles, and that they are built back in headquarters
and are transported to the tentacles by unknown means.

LENHOFF: In the chromatograms you showed, were you run-
ning the entire fluid or a hydrolyzate of the fluid?

LANE: This was the entire material. We took the entire gastro-
vascular fluid without any treatment. I suspect we have amino
acids and peptides. We are now analyzing this fluid using paper
electrophoresis.

LENHOFF : What was the solvent?

LANE : n-propanol.

LENHOFF: In H. littoralis we find that the gastrodermis takes up
mostly particles, and leaves behind the free amino acids in the
gut. Your chromatogram looks somewhat like a normal pattern
of free amino acids. Do you think Physalia does the same thing?

LANE: We'll know more about this very soon. We find that toxin
peptides distribute very much like this and we have eluted, hydro-
lyzed, and rechromatographed them separately. We know they are
peptides. So without actually having done it on this gastrovas-
cular material, I feel fairly certain that these are peptides also.

WOOD: Do you have any real evidence that the gastrodermis
extrudes materials into the mesogleal extracellular space, which
are then picked up by the epidermal cells? I question this because
it seems to me that it would be more efficient to transfer such
materials directly. This bears on whether your specialized area
in the mesoglea is cellular or is purely connective tissue?

LANE: That was the way we had interpreted it, but this is
purely tentative and subject to change. Having seen the way in
which both endodermal and ectodermal processes interdigitate and
weave their way through the mesoglea in hydra, we could easily

CHARLES E. LANE 177

expect the same thing to take place here. It may be that these
tremendously hypertrophied endodermal cells penetrate through the
mesoglea in these regions.

MARTIN: I want to mention an experiment which supports
Dr. Lane's hypothesis. We didn't work with Phy.salia or hydra,
but with Anthopleura elcgantissima. We separated the tentacles
from the column and took the mesenteries out. Then we ground
up the column and tentacles separately, made extracts and mea-
sured their toxicity by injecting them into mice and we found that
the extracts of the nematocyst-poor column was as toxic as a
crown, which is nematocyst-rich.

HAND: Did you remove the mesenterial filaments?

MARTIN: Yes.

HAND: Fine.

MARTIN: By the way, the mesenteric filaments were less toxic
than the other two fractions.

HAND: That's quite contrary to what I would have expected,
since they have the bulk of the internally located nematocysts.

SLAUTTERBACK: There is a mesogleal formation in hydra
somewhat similar to the specialized areas you described. A great
accumulation of mesoglea is sometimes seen under the pedal disk
secretory cells. The predominant component here is amphorous.
Whether it is the same material commonly found in the mesoglea
has not been determined. The fine filaments and glycogen granules
are not increased as much.

Also, is the greatly enlarged part of the hypertrophied gastro-
derm cells an enlarged "central vacuole "?

LANE: Yes.

SLAUTTERBACK: Were you ever able to fix anything in that
vacuole?

LANE : We've tried a wide spectrum of fixatives on these vacuoles
but they've always been clear.

BURNETT: Have you ever found nematocysts in the gastroder-

178 THE BIOLOGY OF HYDRA : 1961

mal cells suggesting that there may be a migration through the
gastrovascular cavity?

LANE: Yes, I've found them in gastrodermal cells, but they
have been in cells which have incorporated this material from
prey organisms.

BURNETT: Are they in a state of digestion?

LANE: That's right. Normally the nematocysts end at the lip of
the gastrozoid. The lip is always identifiable by having cnidoblasts
in its ectoderm, but none in its gastroderm.

Actually, we've had a great deal of trouble in keeping these
animals in captivity. Probably the reason for this is that they have
no protection against dragging their tentacles on the bottom. When-
ever this happens the fragile surface epithelium is destroyed so
that the next time the tentacle contracts, it squirts out some of this
gastrovascular fluid. It's interesting that within an hour of placing
a mature PhysoUa in an ordinary aquarium tank, the surrounding
water becomes ninhydrin-positive. He loses much fluid. This is one
reason why we have been unable to keep these animals in captivity
long enough to feed them, and then study the distribution of
digested food materials to the gastroderm.

LARSON What can you tell us about the pharmacological action
of the toxin?

LANE: We haven't enough information on the pharmacology
of the toxin to justify any statements.

GOREAU: Can rabbits be immunized against Physalia toxin?

LANE: Yes. The material is sufficiently antigenic to develop good
titers. It is difficult, however, to difl^erentiate between a lethal and
an immunizing dose.

GORDEAU: That's the problem of anaphylactic shock which
was discovered with Physalia toxin by Richet. If you could immunize
an animal against the toxin and label the antibodies with suitable
fluorescent groups it might be possible to find out whether there
is transfer of toxin from the gastroderm through the mesoglea into
the epidermal nematocyst batteries.

LANE : Yes, that would be an interesting experiment.

Compounds of Pharmacological
Interest in Coelenterates

John H. Welsh

Biological Laboratories, Harvard University, Cambridge, Massachusetts

The nematocysts of coelenterates appear to serve two principal
functions: one, a means of protection, the other, a role in feeding.
A person once badly stung by Physalia, Cijanca or certain of the
cubomedusae avoids contact with one of these a second time. It
may be assumed that an animal that is stung and survives also may
avoid future contact with a coelenterate if it is capable of learning.
More important, perhaps, to the coelenterates, is the paralyzing or
relaxing action of the contents of the nematocysts when injected
into their prey.

Since the very early years of this century, efforts have been
made to identify the substances in coelenterates that are responsible
for the symptoms that result from their sting. In most of the earlier
work, extracts of whole coelenterates or of nematocyst-bearing
parts (tentacles and acontia) have been used. Therefore, it has not
been possible to attribute an observed action to nematocyst con-
tents. The recently developed methods of isolating clean nemato-
cysts will obviate this difficulty if it can be shown that they lose
none of their contents during the isolation procedure.

A condensed and incomplete summary of substances or frac-
tions obtained from various coelenterates follows. Some of these
derive from nematocysts; others, almost certainly, do not.

I. Early attempts to isolate toxic components of coelenterates by
Richet and Portier (19, 20, 21) yielded three active extracts:
"thallasin," "congestin" and hypnotoxin." None of these was chem-
ically identified and each was doubtless a mixture of substances

179

180 THE BIOLOGY OF HYDRA : 1961

(see refs. 13, 22, 23, 25, for summaries of this and other earHer
work ) .

II. Quarternary ammonimii compomids:

Several nitrogenous bases have been isolated from various coe-

lenterates, including the following: „ .

° ° References

tetramethyl ammonium hydroxide or "tetramine" 4, 11, 17, 27

N-methylpyridinium hydroxide 5

homarine 2, 10, 17, 27

trigonelline 2, 27

y-butyrobetaine 1, 27

zoo-anemonin 3, 6, 27

Of these bases, the only one that has marked paralyzing action
is tetramine (4, 27). It is the only one fomid thus far in a fresh-
water coelenterate (27). It is a known toxic component of certain
molluscan tissues (7, 9). With the exception of zoo-anemonin, the
other bases listed above are widely distributed among marine
invertebrates where they may play a role in osmoregulation ( 10, 27 ) .

III. 5-Hydroxytryptamine (serotonin, 5-HT) :

This very potent pain-producer and histamine releaser has now
been identified in a variety of coelenterates (17, 25, 26). It is pres-
ent in the coelenteric tissues of Calliactis parasitica in very large
amounts ( 17 ) but in other coelenterates it is most abundant in
regions (tentacles and acontia) where nematocysts are concentrated
(25,26).

IV. Histamine and histamine releasers :

Histamine has been found in some coelenterates but not in
others (17, 24). Potent histamine releasers have been extracted
from a sea anemone ( 12) and Cyanea (24) .

V. Active proteins:

Much evidence indicates that the paralyzing and edema-pro-
ducing actions of coelenterate toxins are due, in large measure,
to a protein component(s) (8, 13, 14, 15, 16, 21, 22, 23). There
is some evidence that this component acts on cholinergic neurons

JOHN H. WELSH 181

in such a manner as to block conduction and or transmission
( 17 ) . The neutrahzing action of certain acetylchohne blockers such
as tetraethylammonium (TEA) on the paralyzing action of ten-
tacle extracts supports this view (25).

Certain of the symptoms that follow a coelenterate sting such
as pain, burning, itching, localized edema and hemorrhaging
could result from injected 5-HT (a potent pain producer and his-
tamine releaser), from histamine itself, and from other histamine
releasers. These sul^stances, however, cannot be responsible for
the paralyzing action of the nematocyst contents. Many quaternary
ammonium compounds do have a paralyzing action as junctional
blocking agents. Of those listed above, only tetramine can qualify
as a candidate for the paralyzing action. In the first place it is the
only one that has been identified in hydra extracts, while most
of the others are widely distributed among the marine invertebrates.
In the second place, tetramine is an effective poison and is the
toxic component of the salivary glands of certain marine gastropods
(7, 9), while the others are surprisingly non-toxic (cf. 27). Fur-
thermore, the earlier observed antagonism of coelenterate extracts
by tetraethylammonium chloride or Banthine (25) strongly sug-
gests that a methylated quaternary nitrogen compound is, in some
way, involved in the paralysis resulting from a coelenterate sting.
However, calculations may be made that indicate that there is
not enough tetramine, in the extracts that we have used, to account
for their paralyzing action, at least on arthropods.

Evidence has been accumulating over the years that the paralyz-
ing factor in coelenterate toxins is a protein or group of proteins.
Several recent studies show that toxicity remains after dialysis but
is destroyed by boiling and by treatment with certain proteolytic
enzymes (14, 15, 16, 18). The exact mode of action of the toxic
protein ( s ) is not yet clear.

RfiSUME OF SOME EXPERIMENTS THAT ARE
CURRENTLY IN PROGRESS

We are, at present, comparing the actions of homogenates of
Metridium acontia and whole Hydra, and of material discharged

182 THE BIOLOGY OF HYDRA : 1961

from their nematocysts by electrical stimulation, on Carcinus mae-
nas, Uca ptigilator and several species of cockroaches. A brief
resume of some of the experiments and tentative results follows:

1) The minimum lethal dose of a homogenate of Hydra ameri-
cana, in terms of the number of Hydra injected is between 5 and 10
Hydra for Carcinus weighing 20-30 gms.; 2-3 Hydra for Uca weigh-
ing 4-5 gms.; and about 5 Hydra for female Bryostria sp. (cock-
roach) weighing 4-5 gms. These are doses that usually kill in from
1 to 24 hours. The average dry weight of Hydra amcricaua, reared
in the laboratory, is about 35 [xg. If the paralyzing factor constitutes
something like 0.1% of the total dry weight, it appears that 0.2-0.4
fig. of toxic substance is lethal for a 20-30 gm. green crab.

2) Heating a Hydra homogenate for 30 min. at 100 "^ results in
complete or nearly complete loss of paralyzing action.

3) Electrical stimulation of numbers of Hydra (200-300) in a
minimum volume of distilled water discharges many of the nema-
tocysts. Injection of a small volume (0.05 ml.) of the fluid sur-
rounding the Hydra into Uca, produces symptoms that are quali-
tatively like those seen when whole Hydra homogenate is injected.

4) Hydra, and Mctridiiim acontia, have been homogenized in
1.0% tetraethylammonium chloride (TEA). When volumes are in-
jected known to contain minimum lethal doses of Hydra or acon-
tia, none of the characteristic symptoms develop and most test
animals survive indefinitely. This agrees with earlier observations
on the autotomy reflex in brachyurans when it was found that
TEA very effectively antagonized the effects of coelenterate ex-
tracts (25). If the TEA is blocking the action of a toxic protein
component, and not tetramine only, this may provide a clue to
the mode of action of the toxin.

REFERENCES

1. AcKERMAN, D., 1927. tjber die IdentiUit des Atkinins mit dcm 7-Butyrobetaine.

Zeitschr. f. Biologie, 86: 199-202.

2. AcKERMAN, D., 1953. Uber das Vorkommeu von Homarin, Trisonellin und einer

neuen Base Anemonin in der Anthozoa Ancmonio sulcata. Zeitschr. f.
physiol. Chemie, 295: 1-9.

3. AcKERMAN, D., 1954. Richtigstellung: "Zoo-Anemonin" statt Anemonin. Zeitschr.

f. physiol. Chemie, 296: 286.

JOHN H. WELSH 183

4. AcKERMAN, D., F. HoLTZ, aiid H. Reinwein, 1923. Reindarstellung und Kon-

stitutionsermittelung des Tetramines, eines Giftes aus Aktinia equina.
Zeitschr.f. Biologie, 79: 113-120.

5. AcKEKNiAN, D., F. HoLTZ, and H. Reinwein, 1924: tjber die ExtraktstofFe von

Aktinia equiyui. Zeitschr. f. Biologie, 80: 131-136.

6. AcKERMAN, D., and P. H. List, 1960. Zur Konstitution des Zooanemonins und

des Herbipolins. Zeitschr. f. physiol. Chemie, 318: 281.

7. AsANO, M., and M. Itoh, 1960. Salivary poison of a marine gastropod, Neptunea

arthritica Bemhardi, and the seasonal variation of its toxicity. Ann. N.Y.
Acad. Sci., 90: 674-688.

8. Cantacuzene, J., and A. Damboviceanu, 1934. Caracteres physico-chimiques

du poison des acconties d'Adamsia palliata. C. R. Soc. Biol., Paris, 117:
138-140.

9. Fange, R. 1960. The salivary gland of Neptunea antiqua. Ann. N. Y. Acad. Sci.,

90: 689-694.

10. Gasteiger, E. L., p. S. Haake, and J- A. Gergen, 1960. An investigation of

the di.stribution and function of homarine ( N-methyl picolinic acid). Ann.
N. Y. Acad. Sci., 90: 622-636.

11. Haurowitz, F., and H. Waelsch, 1926. Uber die chemische Zusammensetzung

der Qualle Velella spirans. Zeitschr. f. physiol. Chemie, 161: 330-317.

12. Jacques, R., and M. Schachter, 1954. A sea anenome extract (thalassine) which

liberates histamine and a slow contracting substance. Brit. J. Pharmacol., 9:
49-52.

13. Kaiser, E., and H. Michl, 1958. Die Biochemie der tierischcn Gifte. Franz

Deuticke, Vienna.

14. Lane, C. E. 1960. The toxin of Physalia nematocysts. Ann. N.Y. Acad. Sci. 90:

742-750.

15. Lane, C. E., and E. Dodge, 1958. The to.xicity of Physalia nematocysts. Biol.

Bull, 115: 219.

16. Martrm, E. J., 1960. Observations on the toxic sea anenome, Rhodactis howesii

( Coelenterata ) . Pacific Science, 14: 403-407.

17. Mathias, a. p., D. M. Ross and M. Schachter, 1960. The distribution of

5-hydroxytryptamine, tetramethylammonium, homarine, and other sub-
stances in sea anenomes. /. Physiol., 151: 296-311.

18. Phillips, J. H. Jr., and D. P. Abbott, 1957. Isolation and assay of the

nematocyst toxin of Metridium senile fimhriatum. Biol. Bull., 113: 296-301.

19. RiCHET, C. 1902. Du poison pruritogene et urticant contenu dans les tentacules

d'Actinies. C. R. Soc. Biol, Paris, 54: 1438.

20. RiCHET, C., 1903. Des poisons contenus dans les tentacules des Actinies, con-

gestine et thalassine. C. R. Soc. Biol, Paris, 55: 246.

21. RiCHET, C., and P. Portier, 1936. Recherches sur la toxine des coelenteres et

les phenomenes d' anaphylaxie. Resultats des campagnes scientifiques,
Monaco 95: 3-24.

22. SoNDERHOFF, R., 1936. tJber das Gift der Seeanenionen. I. Ein Beitrag zur

Kenntnis der Nesselgifte. Liehig's Ann., 525: 138-150.

23. Thiel, M. E. 1935. Uber die Wirkung des Nesselgiftes der Quallen auf den

Menschen. Ergebnisse u. Fortschr. der Zoologie, 8: 1-35.

24. UvNAS, B. 1960. Mechanism of action of a histamine-liberating principle in

jellyfish [Cyanea capillata). Ann. N.Y. Acad. Sci., 90: 751-759.

25. Welsh, J. H., 1956. On the nature and action of coelenterate toxins. Deep Sea

Research, 3(suppl): 287-297.

26. Welsh, J. H., 1960. 5-Hydroxytryptamine in coelenterates. Nature, 186: 811.

27. Welsh, J. H., and P. B. Prock, 1958. Quaternary ammonium bases in the

coelenterates. Biol Bull, 115: 551-561.

184 THE BIOLOGY OF HYDRA : 1961

DISCUSSION

HAND: The extra serotonin that you find in the acontia seems
reasonable in view of some very simple observations that one can
make on Metridium and other acontiate anemones. They commonly
eat small worms, copepods, and things of this nature. If you get a
small transparent anemone, you can see that after the food is swal-
lowed the prey is still kicking, wriggling and squirming. It gets into
the coelenteron and the acontium coils around the animal, presum-
ably the nematocysts of the acontium discharge, and this very
quickly subdues it. It quivers a couple of times, and then stops.
The acontia, of course, are rich in nematocysts,

ROSS: Do you think that the amounts of serotonin that you
find in the acontia, ca. 1 Mg./g., is significant? Compared with the
amounts that we found in other parts of anemones, they seem so
small that we would have dismissed them.

WELSH: Well, I think if I may say so, it was unfortunate that
you looked at Calliactis first. I think if you had looked at other
anemones you would have viewed this situation differently.

ROSS: Not at all.

WELSH: Let me put it this way. If you go out and catch a
vicious stinging wasp, you can get out of its venom a perfectly
tremendous amount of serotonin. You measure it as 6 to 20
milligrams per gram of venom. Now if you do its nervous system,
you get a few tenths of a microgram. I believe that the serotonin
in the nervous system is just as important in the life of the wasp
as the serotonin in its venom. The most we have in any part of
our nervous system is 0.4 micrograms per gram of hypothalmus.
And if the tranquilizer reserpine is doing what they say its doing,
releasing serotonin, then this brings this down to a 10th of that,
and here we're working in the lOths and hundredths of micro-
grams per gram range. This is less than the concentration range
of serotonin that one finds in acontia.

ROSS: We found 600 times as much in the lining of the coelen-
teron in Calliactis, so this made us think it couldn't possibly
be associated with nematocyst poisons.

JOHN H. WELSH 185

WELSH: But when you looked at other parts, you found that the
tentacles were richer than the body wall?

ROSS: A bit, but on the borderline.

LENHOFF: Couldn't we view the tetramethylammonium com-
pounds not as toxins, but as part of the normal nervous system trans-
mitters of coelenterates since tetramine is present in all of the tis-
sues assayed? I ask this question because when glutathione activates
the feeding response in H. littoralis, some of the few substances
that enhance the response are certain tetramethylammonium com-
pounds. Possibly the transmission of the glutathione stimulus goes
through a tetramine-mediated pathway rather than through an
acetylchloline-mediated pathway?

WELSH: I think it is entirely possible. We have no evidence on
the tetramine one way or the other. However, tetramine does occur
in a number of venoms; it occurs in the salivary glands of some
marine gastropods in large amounts. And, of course, other choline
esters, and other quaternary ammonium compounds occur in certain
molluscs. But that tetramine may be taking the place of acetylcho-
line in the coelenterate nervous system is a good possibility.

HESS: Do these animals have choline esterase or acetylcholine?

WELSH: There is choline esterase.

PASSANO: I suspect that the acetylcholine esterase system is
not significant in the functioning of the scyphozoan nervous sys-
tem, and we know that 5-hydroxytryptamine also fails to have
any effect. Could it be that the use of these substances, toxic to
other animals as nematocyst toxins, might be valuable to the
coelenterates because they would avoid the danger of self-inHicted
paralysis? Is this even why their neuropharmacology is different
from that of other animals?

WELSH: Venomous animals are generally successful in keeping
their venoms away from themselves.

PASSANO: Well I would like to ask then, in other people^s
experience in studying the feeding responses of nematocyst-bear-
ing animals, are the nematocysts always prevented from penetrating

186 THE BIOLOGY OF HYDRA : 1961

the animal that possesses them? The independent effector is quite
different from the effector at the end of a wasp; it is not so neatly
controlled. The tentacle of a coelenterate coils around its prey.
There is a great chance for nematocysts to be discharged into a
tentacle. This would obviously create difficulties if the tentacle was
paralyzed by its own poison.

BURNETT: It is common for hydra to pierce its own tissues
with nematocysts during feeding.

PHILLIPS: I think the experiments of Dr. Ross, and of Dr.
Martin and the ones that I did on Metridium suggest that caution
should be employed in the interpretation of work using whole
tissue extracts. Sometime ago, when I was working on the toxin,
I detected a 5-hydroxyindole compound, which at that time I
thought corresponded more closely to bufotenin. On purification
I noticed that the level of 5-hydroxyindole compounds decreased
steadily. In fact, pure suspensions of nematocysts contained no
detectable 5-hydroxyindole compounds at all, yet the nematocysts
were still capable of discharging and still possessed toxicity.

WELSH: In that connection, I would be interested to know if the
5-hydroxytryptamine washed out of the nematocyst. It's a small,
soluble molecule that diffuses readily through some cell surfaces.

PHILLIPS: This is a possibility. But the nematocyst suspensions
after purification still should show toxicity.

WELSH: I don't think that the serotonin is really toxic. You
can put a large amount of serotonin into a crab and it gets very
nervous and jittery. An hour later it is normal.

PHILLIPS: Diffusion from the nematocyst during purification, of
course, is always a possibility. At the same time, nematocysts are
still susceptible to osmotic discharge, so that gross permeability
changes do not seem to have occurred.

LOOMIS: How do you keep your nematocysts from discharging
while you separate them?

PHILLIPS: With high concentrations of sucrose.

Present State of Nematocyst

Research: Types, Structure

and Function

Cadet Hand

Department of Zoology, University of California, Berkeley, CalifomiOi.

I want to start by quoting an admirable passage from the Intro-
duction of the recent paper by Burnett and colleagues (1). On
page 247 they state "One of the most structurally complex and cer-
tainly one of the most enigmatic organelles in the animal kingdom
is the nematocyst of coelenterates. For nearly a century hosts of
scientists, too numerous to mention, have concentrated their at-
tentions on the mode of formation, the migration pathways, the
mechanism of discharge, and the chemical nature of these unusual
structures. . . ." These same authors go on to make the statement
that ". . . none of these subjects of investigation has been resolved to
any degree of satisfaction." In many ways this statement is accu-
rate and acceptable, but I think in many ways I would disagree with
the generality. A good deal is known about each of the subjects
they cite and I for one have found considerable satisfaction in the
numerous papers on nematocysts that I have examined. I also want
to acknowledge that some of my satisfaction has come from reading
the papers of Burnett and his co-workers.

I want to talk today about types, structure and function of nema-
tocysts. I also want to make it clear that I do not work on nemato-
cysts, I work with them. My interests in them are twofold. First,
nematocysts are a truly \'aluable systematic tool and many coelen-
terates can be positively identified by their nematocysts alone. Not
only this, but nematocysts are useful in relating higher taxa such

187

188 THE BIOLOGY OF HYDRA : 1961

as genera, families or even orders, and in the broad view even
classes. Second, as a student of coelenterates I am interested in the
biology of these animals, and the nematocysts are intimately in-
volved in numerous aspects of the lives of coelenterates.

There have been several attempts to classify nematocysts and
some of the results of these have come down to us in the form
of such useful and descriptive names as penetrants and glutinants.
However, it was not until the elaborate system of Weill (10) was
published that any real uniformity of nomenclature of nematocysts
was arrived at. With the introduction of Weill's terminology some
people complained that the system was too clumsy and the names
too long to be useful. For example the commonest penetrant of
many anthozoans could be called a hoplotelic microbasic masti-
gophoric rhabdoidic heteronemic stomocnidic nematocyst, or a
stenotele could be called a stenotelic rhopaloidic heteronemic
stomocnidic nematocyst. In common practice, and as Weill's termi-
nology is being applied, the names microbasic mastigophore (or
just mastigophore) and stenotele suffice. Weill's system is only for-
bidding when one first meets it, but it is a defined system which
makes possible far greater accuracy in communication than any
other so far devised. To use the full nomenclature, as in the exam-
ples I cited, is just as absurd as to start the name of some species
with the phylum name, add in the names of the class, order and
family and finally tack on the specific binomial.

Weill's system recognizes two categories of cnidae, spirocysts
and proper nematocysts. Spirocysts are restricted to the zoantharian
anthozoans while all coelenterates have nematocysts. The struc-
ture and function of spirocysts are obscure. Weill (10) believes that
spirocysts have but a single layered wall and it is extremely rare
to see a spirocyst which has everted its thread. Cutress (5) has ar-
gued rather convincingly that spirocysts are nematocysts and from
his comments one could conclude that they represent a form of
holotrichous nematocyst. The test of this conclusion will undoubt-
edly come when a study of these cnidae is carried out with an
electron microscope.

The nematocysts proper have two major subdivisions, astomo-
cnidae whose tubes are closed and stomocnidae with tubes open at
the tip. The astomocnidae are divided in turn into two categories.

CADET HAND 189

the familiar desmonemcs or volvents, and the much less familiar
acrophores and anacwphorcs of the Siphonophora, which are col-
lectively called rhopalonemcs and have a sac-like tube rather than
the coiled or corkscrew tube of the desmonemcs.

The stomocnidae show much more variety in form. They can be
divided into the haploncmcs, whose tube has no enlarged basal
portion or butt, and the hcteroncmcs which have a butt. Among the
haplonemes we find the familiar armed holotrichs and unarmed
atrichs, as well as partially armed forms we call basitrichs. These
haplonemes have a thread or tube of constant diameter and are
technically isorhizic. A second type of haploneme has an aniso-
diametric tube which may taper or be slightly swollen near the base.
These are the anisorhizic nematocysts of various siphonophores and
Tubularia.

The heteronemes, which you recall have a butt, can be divided
into the rhahdoidcs whose butts are isodiametric and the rhopa-
loides whose butts are anisodiametric. The rhabdoides can in turn
be subdivided into masiifiophores with a terminal thread and
amastigophores which have no terminal thread, while the rhopa-
loides may be subdivided into eurijtclcs whose butts are dilated at
their distal ends and stenoteles whose butts are dilated at their bases.

Further subdivisions of a number of the nematocyst categories
mentioned abo\e were proposed by Weill (10) but it is not neces-
sary to review them further here. Weill's system described a total of
eighteen different nematocyst categories, and in fact made it possi-
ble by applying the terms hoplotelic for armed threads and anaplo-
telic for unarmed threads to distinguish two sub-types within most
of the subdivisions of the heteronemes.

Working from Weill's system still other kinds of nematocysts
have been described. Carlgren (2) divided mastigophores into
b-mastigophores and p-mastigophores, based on the appearance of
the end of the inverted butt. The p-mastigophore was the type Weill
(10) had described and the b-mastigophore was a new category
which in its unexploded condition looked like a basitrich but when
exploded looked like a mastigophore.

Another worker, Cutress (5), using the light microscope de-
scribed two further categories of nematocysts, q-mastigophores and
macrobasic p-mastigophorcs, and proposed the elimination of

190 THE BIOLOGY OF HYDRA : 1961

amastigophores (microbasic and macrobasic amastigophores of
Weill). Ciitress also made a number of claims about nematocysts,
some of which are wrong and others certainly are questionable.
Unfortunately we have not yet progressed far enough in our study
to analyze critically all of the structural details of all nematocysts,
and until electron microscope studies have been extended to many
more types of nematocysts, a number of suggestions Cutress has
made cannot be proven or disproven.

One of Cutress' suggestions is that the shaft or butt of mastigo-
phores is folded within itself as well as being inverted before ex-
plosion. This would bring the point of the butt to the tip of the cap-
sule, would keep the point in the lead as the basal half of the butt
everts, and he claims the thread is attached to this leading tip of the
shaft. The tread would evert after the shaft has completely emerged.
Miss Jane Westfall of the Department of Zoology at the University
of California at Berkeley has been examining a number of nemato-
cyst types with the electron microscope and has been particularly
interested in mastigophores. Her studies have not yet progressed to
a point where publication seems warranted, but we can comment
on Cutress' suggestion. Both cross and longitudinal thin sections
have been examined as well as whole exploded nematocysts. The
material has been primarily the nematocysts of the acontia of our
West Coast Metridiiim senile fimbriatum. Cross sections of micro-
basic amastigophores, microbasic b-mastigophores and basitrichs
(sensu Weill and Carlgren, refs. 2, 10) show clearly that the shaft
is not folded on itself and contains only the spines. The spines are
blades, as was shown so clearly by Robson (8), and are oriented
with their tips toward the open end of the capsule. Longitudinal
sections of amastigophores also show that the notch seen in the
light microscope at the distal end of the shaft of amastigophores and
p-mastigophores is the result of this being the end of the armored
region of the shaft. Moreover, there is no thread within the shaft
as Cutress has proposed. From these observations we conclude that
Cutress is wrong, as were certain earlier workers who proposed
folded as well as inverted shafts. It also should be noted that from
the work of Picken ( 7 ) and Robson ( 8 ) that Cutress' claim that the
holotrichs of Corynactis, which he calls macrobasic p-mastigo-
phores, has an inverted and folded shaft is wrong.

CADET HAND 191

The proposal of Cutress (5) to eliminate the categories micro-
basic and macrobasic amastigophores also is not acceptable. It is
true that there frequently is a short thread on many amastigophores,
but this thread is apparently sometimes entirely absent. In our elec-
tron microscope studies we have failed to find more than a wisp
of a thread at the end of the shaft of these nematocysts in Metri-
dium and in thin sections we have not been able to verify, as Cutress
suggested, that this thread is attached to the inner capsular wall
near the end of the shaft. Studies such as Cutress', which were
based on the light microscope alone, cannot resolve problems such
as this and we must await definitive electron microscope studies.

The new category of nematocysts, microbasic q-mastigophores,
which Cutress described may indeed be a valid type although this
too is open to question. I and other workers have noticed dart-
like structures which characterize q-mastigophores lying among
exploded nematocysts. Weill (10) reports a number of such oc-
currences and reviews some older accounts. These darts, which Cut-
ress says are unattached discrete structures, occur within the shafts
of certain microbasic mastigophores of acontiate anemones. Cutress
reports them from the genera Metridiwn and Aiptasia, to which
I can add Diadumene. It was my conclusion that the darts in
Diadiimenc franciscana were nothing more than the mass of spines
which should have armed the shaft. These spines are tightly curled
within the shaft as we have seen in electron micrographs (unpub-
lished) and are commonly sloughed off soon after eversion of the
shaft as many workers have noted. Little would be required for this
mass of spines to stick together, lose their contact with the shaft and
form the dart. Whether this happens accidentally or as a nonnal
process is not known. In Diadumene franciseana the darts could
usually be found lying near a mastigophore with no spination on
the shaft. Cutress, however, figures darts emerging from mastigo-
phores with spined shafts and associated with nematocysts with
spined shafts. If these are accurate observations, the recognition of
a special nematocyst, the microbasic q-mastigophore, certainly is
called for. It is unfortunate that Cutress did not choose some other
name than dart for the organized structure contained in his q-masti-
gophores. This name, dart, had already been used by Picken (7)
to describe the tip of the packed spines as they emerge from the

192 THE BIOLOGY OF HYDRA : 1961

everting thread. Both structures would appear to be for penetration,
and both may be the same if Cutress' interpretation is wrong. If,
however, Cutress is correct two things would seem apparent. First
with such a large structure as Cutress' dart seems to be, the spines
of his mastigophore cannot be as large as those figured by Robson
(8) nor as seen in Miss Westfall's micrographs because there would
not be space for both. In Miss Westfall's unpublished electron
micrographs the spines completely fill the shaft, and Cutress
figures spines which would appear to be normal, at least as we see
them in the light microscope (see ref. 5, p. 132, Fig. 7b and c).
Second, it will continue to be confusing if two dissimilar parts
of nematocysts have the same name and Picken's use of the word
dart has priority.

Another difficult point in Cutress' work concerns basitrichs. It is
his contention that the category of nematocysts Weill ( 10 ) identi-
fied and defined as basitrichs are in fact for the most part better
assigned to the category microbasic b-mastigophore. Cutress is
correct when he notes the difficulty in solving the problem with the
light microscope because the basic problem here is to determine
whether one is dealing with isodiametric isorhizas or with hete-
ronemes with a butt. The magnitude of the difference between butt
and thread may be as little as 0.1 microns Cutress notes, and this
is not a readily resolvable difference with a light microscope. Cut-
ress solves the problem by arbitrarily deciding that when one sees
a straight inverted shaft, as in Weill's basitrich, this means the tube
of this portion is differentiated as a shaft, is greater in diameter
than the thread and that the tube itself is stilfer than the thread.
The fact that this portion of the tube, the straight part carrying the
armature, may be stiffened and not coiled only because it is packed
with spines seems not to have occurred to Cutress. Cutress suggests
we restrict basitrichs to certain nematocysts which so far are known
only from anthozoans and have no stiffened or straight part in the
inverted tube. These nematocysts, as he shows in his Figure 3, are
basitrichs in every sense. In our electron microscope work we have
examined uneverted basitrichs. The wall of the spined portion is
not thicker than the wall of the thread. We cannot comment on
diametric relationships since it would be the everted, not uneverted
picture which should be examined and we have not done this. These

CADET HAND 193

basitrichs look structurally very much like the much larger micro-
basic b-mastigophores we have looked at in the same tissue, the
acontia of Metridium. Cutress may be correct in writing "It may be
presumptuous to state that the man who defined almost the entire
system of cnidae classification failed to recognize his own categories"
(ref. 5, page 126), but it seems "presumptuous" to me for Cutress to
have done this on what appears to be spurious logic which assumes
a shaft, rather than on factual evidence such as the electron micro-
scope could have produced. At any rate, the evidence is not in yet
and whether most basitrichs, as we have known them from the liter-
ature, are in fact b-mastigophores remains to be seen. If Cutress is
correct the identification of microbasic b-mastigophores will be
much easier than it is today.

My last comments on Cutress concern his new category of macro-
basic p-mastigophores. By definition this category is said to have
the undischarged shaft inverted and folded back on itself. This
certainly is not so as I noted earlier, nor do I believe that this cate-
gory includes the holotrichs of Corynactis as Cutress states. In our
Corynactis caJifornica the holotrichs are good isorhizas, that is the
thread is isodiametric. The category Cutress proposes would in-
clude the former macrobasic amastigophores, and again I would say
that the shortness of the thread, if it exists at all, is good reason for
keeping the amastigophore separate from the p-mastigophore. It
also seems reasonable that macrobasic p-mastigophores do exist,
but they differ strikingly in their appearance from the microbasic
p-mastigophore which has the obvious long coiled terminal thread
within the capsule.

The comments I have made so far concern both structure and
types of nematocysts and I do not intend to review the details of
fine structure which are so well known to so many and which we
are adding to almost daily as new electron micrographs are exam-
ined. The work of Chapman and Tilney (3, 4) stands out as
the best work to date on the fine structure of fully formed nemato-
cysts, and the work of Slautterback and Fawcett (9) on the de-
velopment of nematocysts is clearly the best on this subject to this
date. That this elegant work is being done on hydra is little wonder
when one considers how easy this beast is to handle in the labora-
tory, primarily as a result of Loomis' studies. What are needed are

194 THE BIOLOGY OF HYDRA : 1961

studies of many different coelenteiates so that all of the types may
be fully explored rather than merely the limited cnidom of hydra.

I would like to briefly explore one other aspect of nematocysts,
namely their function and functioning. We have not yet arrived at
a point where any single explanation can be had as to how a nema-
tocyst discharges nor do we understand the meaning of diversity in
nematocysts. Diversity in some microscopic structures such as lepi-
dopteran scales and perhaps some sponge spicules seems not to
be adaptive. This is, they all perform the same function and as
long as a given size or distribution is maintained, variation in shape
and ornamentation apparently can occur without selective forces
coming into action.

In nematocysts we do know that some of the diversity is adap-
tive. There is little doubt as to the role of stenoteles and desmonemes
in hydra and the recent work of Burnett, Lentz and Warren ( 1 )
has shown that the desmonemes respond before the stenoteles, trap
the prey and hold it till the stenoteles discharge and kill it. Also,
it appears clear from the work of Ewer ( 6 ) that the atrichs discharge
against smooth surfaces and presumably are sticky, or are gluti-
nants. Ewer also showed that foodstuffs or extracts from food in-
hibited the atrichs while enhancing the discharge of stenoteles. Any-
one who has worked with nematocysts has soon discovered that not
all types respond to all stimuli, and some types like atrichs and spiro-
cysts are very difficult to discharge under most conditions. How-
ever, with all the work that has gone on we still can identify only
three functions for nematocysts as far as the biology of the animal
is concerned, namely adhering, entangling and penetrating, al-
though Ewer ( 6 ) has suggested that the holotrichs of hydra may be
purely defensive. The penetrating types are all assumed to deliver
toxins and poison to the prey or foe but this has not been proven.
We have no described or specific function for most nematocyst
types and in fact our knowledge is limited in that what is known
about function comes entirely from hydra. The work on the nema-
tocysts of other types of coelenterates has concerned itself with
biochemical problems, with studies of discharge mechanisms, the
toxins and the makeup of the capsule rather than the function of
the many varied types. Thus we are left with about twenty types of
nematocyst of which we known the function of three. It would ap-

CADET HAND 195

pear that all the heteronemic stomocnidae are penetrants, but the
functions of most haplonemic stomocnidae are not known though
we may assume they are adhesive. Among the astomocnidae we
find the entangling desmonemes, but what of the rhopalonemes?
As well as being in doubt of the function of most nematocysts we
are again faced with diversity for which it is not easy to see adap-
tive values. Cleverly contrived experiments may be able to answer
many of these questions, but the possibility exists that nematocysts
may be another example of variation without functional significance.
At the moment it is difficult for me to imagine what functional
differences one could ascribe to a series of mastigophores with no
threads, short threads or long threads. Such variations exist, how-
ever, and in discrete places and patterns, that is one species
may have one type in one tissue, another in some other tissue, while
a second species will show only one type in one place. Certainly
types deserves attention.

The problem of how nematocysts discharge is a complicated
one and one to which many authors have addressed themselves.
The cnidocil, which is so characteristic of at least some nemato-
cysts of hydra, is not known to be associated with most nemato-
cyst types, and in fact has been reported only in hydrozoans. When
and if a final relationship between cnidocil and discharge in
stenoteles and desmonemes is worked out we still will have to re-
solve the problem of how other nematocysts are related to what is
found here. We have seen no signs of cnidocils in Metriditim acontia.

What the operculum is, or even if it exists in most nematocysts
is a difficult problem. There seems to be little doubt that some sort
of a plug or structure exists at the point on a capsule where the
thread or tube starts everting. In stenoteles the operculum is a real
structure as demonstrated by the electron microscope studies of
Chapman and Tilney (4). In Miss Westfall's studies of nemato-
cysts no operculum has yet been seen, although the material has
not made optimum observation on this point possible to date.

The mechanism of discharge has been analyzed by many people
and I do not feel a detailed summary is called for here. The
recent summary of Chapman and Tilney (3) cites the conclusions
of the various authors and I would single out the reports of Picken
(7) and Robson (8) as those which are most significant. New

196 THE BIOLOGY OF HYDRA : 1961

information will be added as we gather more information on fine
structure and as further chemical and biochemical studies are car-
ried out. One could suggest from observations on available elec-
tron micrographs such as those of Yanagita and Wada (11), Chap-
man and Tilney (4) and unpublished ones of Miss Westfall's that
the shaft of some heteronemes is folded accordion style. If to this
we add the fact that the capsule contracts on explosion, we could
imagine that the shaft of these nematocysts unfolds as it everts.
This may account for the full eversion of heavily armed shafts and
only later would uptake of water play a role in eversion. This sug-
gestion can be at least partly tested by critical analysis of the length
of the sculptured or folded outline of uneverted shafts as com-
pared with the full length of everted ones.

It is a rare field of biology where one can say the last word has
been said and one wonders if such a field exists, but the study of
nematocysts seems clearly to be in its infancy and there is little
chance of running out of problems ( or words ) . I do feel, however,
that with the renewed interest in these intriguing and complicated
structures which has appeared in recent years there is high hope
that many of our problems will be solved. I look forward with ex-
citement to the time in the future when we have enough knowl-
edge to talk about the types, structure and function of nematocysts
rather than what is not known.

REFERENCES

1. Burnett, A. L., T. Lentz and M. Warren, 1960. The nematocysts of hydra

( Part I ) . The question of control of nematocyst discharge reaction by
fully fed hydra. Ann. Soc. Royal Zool. Belgique 90: 247-267.

2. Carlgren, O. 1940. A contribution to the knowledge of structure and distribu-

tion of cnidae in the Anthozoa. Kungl. Ftjmig. Sdllskapets Handl. N. F. 51 :
1-62.

3. Chapman, G. B. and L. G. Tilney, 1959. Cytological studies of the nemato-

cysts of Hydra. I. Desmonemes, isorhizas, cnidocils, and supporting struc-
tures. /. Biophijsic. and Biochem. Cijtol. .5: 69-78.

4. Chapman, G. B. and L. G. Tilney, 1959. Cytological studies of the nemato-

cysts of Hydra. II. The Stenoteles. /. Biophysic. and Biochem. Cytol.
5: 79-84.

5. Cutress, C. 1955. An interpretation of the structure and distribution of cnidae

in the Anthozoa. Systematic Zoology 4: 120-137.

CADET HAND 197

6. Ewer, R. F. 1947. On the functions and mode of action of the nematocysts of

Hydra. Proc. Zool. Soc. London 117: 365-376.

7. PiCKEN, L. E. R. 1953. A note on the nematocysts of Conjnactis viridis. Quart.

Jour. Micros. Sci. 94: 203-227.

8. RoBSON, E. A. 1953. Nematocysts of Corynactis: The Activity of the filament

during discharge. Quart. Jour. Micros. Sci. 94: 229-235.

9. Slautterback, D. L. and D. W. Fawcett, 1959. The development of the cnido-

blasts of Hydra. An electron microscope study of cell differentiation. /.
Biophysic. and Biochem. Cytol. 5: 441-452.

10. Weill, R. 1934. Contributions a I'etude des Cnidaires et de leur Nematocystes.

Trav. Stat. Zool. d. Wimereux Tome 10, 11. Paris.

11. Yanagita, T. M. and T. Wada, 1959. Physiological mechanism of nematocyst

responses in sea-anenome VI. A note on the microscopical structure of
acontium, with special reference to the situation of cnidae within its sur-
face. Cytologia 24: 81-97.

DISCUSSION

GOREAU: To those of us who swim in reefs and sometimes
come into painful contact with Millepora complanata and similar
stinging species, it would be of interest to know what nematocysts
produce the burning sensation and the erythema.

HAND: Four categories have been described: atrichs, basitrichs,
macrobasic mastigophores and stenoteles. One could guess that the
stenoteles and macrobasic mastigophores give you the kick.

MUSCATINE: Has anyone observed the extrusion of substances
from the end of nematocyst threads?

HAND: Yes, I think there is a lot of information about material
being extruded, and one of the places this is most readily visible
is in the big holotrichs that corallimorpharian anemones and some
corals have. First, there is an uptake of methylene blue. Then there
is eversion of the thread as Picken and Robson have explained so
beautifully. And then real droplets of the material leave the terminal
end of the thread. One can see this happening in a fresh prepara-
tion. Whether or not this is the toxin, and what relation this has to
the total picture, is not at aU clear. But certainly there is something
leaving the capsule. And the total volume of the everted system is
in general greater than the uneverted system. In order to create
this, something has had to move into the system or expand within it.

198

THE BIOLOGY OF HYDRA : 1961

ROSS:^ I would like to report some work which is partly on the
point of Dr. Hand's talk.

By chance, a few months ago, I stumbled on a phenomenon
that I think has some bearing on the specialized function that certain
nematocysts can perform. The sea anemone, Calliocfis parasitica,
which I mentioned earlier today, lives on shells of hermit-crabs in
British and Mediterranean waters. About 2 years ago I found that
the anemone gets on the shell by a rather interesting behavior
pattern ( Fig. 1 ) . It will transfer from another surface to the shell by
a maneuver which begins with the adhesion of the tentacles to the
shell; subsequently the animal detaches the pedal disc which then

Fig. 1. Calliactis parasitica adhering to shell by tentacles and (a) detach-
ing pedal disc from plastic plate and (b) swinging detached pedal disc over
towards shell for eventual settling. 4 min. between (a) and (b).

(From Ross, D.M. 1960. Proc. Zoo/. Soc. London, 134: 43-57. Reprinted
by the courtesy of the Society)

swings over and settles on the shell (Ross, D.M. (1960). Pwc.
zool. Soc. Lond. 134: 43-57). But the important point to which I
wish to draw attention is this initial response of the tentacles when

iDr. Donald Ross, Department of Zoology, University of Alberta, Edmonton, Alberta,
Canada.

CADET HAND 199

they adhere to the shell. A few months ago, working at Banyuls
on the French Mediterranean Coast, D. Davenport, L. Sutton and
I looked at this phenomenon and satisfied ourselves that this initial
sticking of the tentacles was due to the discharge of nematocysts
(Davenport, D., D. M. Ross, and L. Sutton. 1961. Vie et Milieu, in
the press). I don't know what kind of nematocyst was involved so
I can't add anything about particular nematocyst types and their
functions, but certainly it was a nematocyst response to the shell.
Now that raises a puzzling point, because these tentacles of Calli-
actis stick very readily to shells when the anemone itself is not on a
shell; but if you pass a shell over the tentacles of a Calliactis that
is already on a shell, its tentacles do not stick. In other words, these
nematocysts seem, at any rate from this first observation, to be
affected b\' whether the anemone's foot is on the shell or not.

We did some experiments to extend this observation. We had
20 Calliactis; 10 of them were settled on shells and 10 were lying
unattached on the floor of a tank. By taking a test shell and touch-
ing it to single tentacles around the disc, one can get a score of the
number of tentacles that stick. When the anemones are on the shell,
one gets a score of the order of 5 or less "tentacle-sticks" in 100 shell-
tentacle contacts. With the animals lying prone in the tank, one gets
a score of the order of 50 or more "tentacle-sticks" using the same
shell. In our experiments we transferred these same animals, al-
lowing those that had been unattached to settle on shells and strip-
ping off those that were attached so that the experiment could be
done in reverse. And then we got a good reversal of the scores;
the animals which were now on shells, which when unattached had
given scores of the order of 50, had now dropped to 5 or less, and
the other group, which when attached had given scores of 5 or less,
had now climbed up to about 50 "tentacle-sticks" per 100. To my
mind this phenomenon raises a crucial point as to whether nemato-
cysts are independent effectors or not. I say this because the only
change made in the experiment is that in one case the anemone has
its pedal disc attached to the shell, and in the other case the pedal
disc is free and unattached. So this observation forces one to con-
clude that the threshold for this kind of nematocyst discharge could
be affected by some form of remote control which in this case
seemed to originate in the pedal disc.

200 THE BIOLOGY OF HYDRA : 1961

HAND: I'd like first just to applaud this work and say this is
exactly what I was asking for, except that you must find out what
these nematocysts are!

CROWELL: About how long did you wait before you retested?

ROSS: A few hours. The anenomes, when you strip them off,
take at least an hour to open up and relax. The other anemones will
also take about an hour to settle securely on the shells. One has to
wait until all are open and all are settled. So several hours always
elapsed between the two sets of observations in our reversal experi-
ments. But we did several such experiments, and each time ob-
tained clear evidence of big differences in the threshold of nema-
tocyst discharge as measured by "stickiness" of tentacles to shell.

GOREAU: There is a matter which may be important in connec-
tion with what Ross just said. Not too long ago we observed at a
depth of about 70 feet a large anemone, probably Bartholomea
annuhta, which has living amongst its tentacles a small red fish and
several shrimp of the genus Periclimcnses. This shrimp moves
freely amongst the tentacles, climbs around on them or hovers just
in front of them, waiting for small fish to come along. As soon as
a fish is in position, the shrimp climbs aboard and proceeds to
remove ectoparasites from the head and mouth. Once finished with
the job, the shrimp returns to its host anemone. Neither the shrimp
nor the commensal fish living among these tentacles excite any
sort of feeding reflex on the part of the anenome. The questions I'd
like to ask are these: "What protects these commensals against the
nematocysts of the host anemone? Do the nematocysts fail to dis-
charge into the animals at all, or are they immune to the action of
the nematocysts? " The observations made by Ross seem to indicate
that there is complete failure to discharge any menatocysts. In other
words, commensal animals living among the tentacles of anemones
can probably do so because they somehow inhibit nematocysts
discharge and do not trigger off any sort of feeding reflex. That's
the thing I don't understand, because I know that such anemones
react instantly to any bits of meat dropped on the tentacles. This
immediately sets off a feeding reaction resulting in flexion of the
tentacles and opening of the stomodeum.

CADET HAND 201

ROSS: From my experience, I think this is a failure of the nemato-
cysts to discharge. Anemones are usually very active when they
are responding to chemical stimuli and discharging their nemato-
cysts. If nematocysts were being discharged, in this case, one would
expect signs of this in the anemone's behavior.

HAND: Davenport and Norris (Biol Bull. 115, 1958) working
with the anenome Stoichactis and the fish Amphiprion, which I be-
lieve were Philippine in origin, concluded that the nematocysts were
not discharging. When a single scale was removed, however, then
the fish gets it fast. As soon as the mucous layer is broken the
nematocysts respond and the animal is in trouble.

MARTIN: In the experiment which Dr. Ross described, I won-
der if you are sure if the reaction of stickiness is a virtue of the
nematocysts or of the epithelium of the tentacles?

ROSS: We managed to induce a nematocyst discharge by offering
small pieces of shell to tentacles, and observing under the binocu-
lars that a large number of nematocyst threads were attached to the
piece of shell. We also found that nematocysts were discharged
into "Cutex " impregnated with tiny shell fragments, but not into
"Cutex" alone used as a control. We were satisfied that it was a
nematocyst discharge when we witnessed the following pheneome-
non: You can present a shell to a Calliactis by bringing it up very
carefully to a single tentacle. If it sticks, that tentacle adheres so
strongly at the tip that you can lift up the whole animal by lifting
the shell. It is impressive to see one of these large anemones hang-
ing from the shell and attached only by the tip of a single tentacle.
I cannot conceive of anything other than a powerful local nemato-
cyst discharge that could produce this particular effect.

MUSCATINE: Have you ruled out a mucous adhesive?

ROSS: We satisfied ourselves by direct observation that they were
not adhering by mucous strands, but that the tentacle was sticking
directly to the shell at definite points of contact and not over the
whole surface.

BURNETT: Perhaps your animals which did not adhere to the
shell were still discharging nematocysts? We have found that satiat-

202 THE BIOLOGY OF HYDRA : 1961

ed hydra still discharge nematocysts when an Artemia strikes the
tentacle, but the nematocyst is quickly released from the tissues of
the hydra and the AHemia falls to the bottom of the culture dish.
If satiated with food, the hydra makes no effort to hold its prey.
Perhaps in your experiments, the nematocysts discharged but were
not retained by the cnidoblasts.

ROSS: I only refer to the original observation which was that
when an animal is on a shell you can brush another shell across it
and there is not the faintest sign of a response. The tentacles are
just brushed aside; they don't stick to it in any way. Yet you have
this phenomenal behavior which is elicited when the animal is off
a shell; it practically pounces on the test shell. Starting from that
observation, we went on and did this other experiment. I wouldn't
say that this is the complete answer, but I think it raises the whole
question of nematocyst control \ ery sharply, even though more in-
vestigation is required to clear it up. You certainly have a very
different type of behavior depending whether the animal's foot is
on a shell or not. It seems to us that this bcha\ ior, when it occurs,
begins with nematocyst discharge.

LOOM IS: Do you think it might be a matter of the shell trans-
mitting calcium to the tentacle and making it sticky?

ROSS: I've tried a good many models of shells and also shells
boiled in alkali to remove organic material. The anemone does not
respond to these; cleaning the shells destroys the activity. If you
present CaUiactis with a perfect plaster of Paris model of the shell,
it shows no interest. The rest of the story (I haven't time for the
evidence here ) is that some substance in the mollusc shell, and not
derived from the crab but from the mollusc, triggers the nematocyst
discharge and the subsequent behavior pattern. It is not responding
to the calcium of the shell, or to any other inorganic constituent, or
the characteristically sculptured surface of the shell. In fact, the
anemone gets on the shell occupied by the hermit-crab by respond-
ing to the ghost of the long-dead mollusc that used to live there. It
has nothing to do with the crab as such.

SLAUTTERRACK: Does anyone care to go into metaphysics
further? If not, I declare this meeting adjourned.

Activation of the Feeding
Reflex in Hydra lift oralis

HowAiiD M. Lenhoff

Laboidtories- uf Biochemist nj, Howard Hiiiilies Medical Institute, and 7.oology
Department, University of Miami, Mianii, Florida

Throughout tliis talk, I will often speak of experimenting on
Hydra as if these animals were systems of purified enzymes. I
speak in these terms more eonfidently toda\' than I could have a few-
years ago when I first tried to adapt my former training in enzym-
ology to experimentation with live Hydra. In enzymology I was
able to treat a relatively simple experimental system in a limited
number of ways, and the results were usualh' clear and unambig-
uous. I soon found, however, that Hydra could be treated in vir-
tually an unlimited number of ways and that the measurable
responses of the animal were more difficult to interpret correctly.

During a rewarding apprenticeship with Dr. Loomis, I was
introduced to his method for rearing Hydra in the laboratory in
solutions of known composition (16), a development that has en-
abled inxestigators to experiment with hydra using the same rigor-
ous controlled conditions which are applicable to simpler systems.
These first discoveries of Loomis opened the door wide to contem-
porary hydra research.

His selection of Hydra for use in quantitative studies of cellular
problems was a happy one because of at least three intrinsic prop-
erties of the animal. First, genotypic constancy is practically guar-
anteed by using animals descended from a single individual by
budding. Second, their small size and lack of skeleton lend
them to many of the quantitative techniques (7, 14) applicable
to simpler systems. But perhaps the feature of hydra which makes

20.3

204 THE BIOLOGY OF HYDRA : 1961

them so remarkably adaptable to quantitative study is their lack
of a definite self-regulated internal extra-cellular fluid. In place
of this fluid is their culture solution, a solution regulated by
the investigator. Once the environment is controlled, individual
variation between hydra is minimized and thus the results are
rendered less ambiguous.

Working on the assumption that the intact Hydra can be treated
with the same controlled conditions that we normally employ with
an enzyme in solution, we find that in order to get reliable results
with the glutathione-//j/f/rfl system, we must control precisely and
within restricted limits the following factors, some of which I will
report on today: pH, nature of the buffer, ionic strength, the nature
of both the cations and anions, temperature, presence of trace
metals, amount of aeration, concentration of glutathione or related
compounds, presence or absence of proteases or glutamic acid, and
length of time since previous exposure to glutathione or since last
feeding. Undoubtedly, there are other factors that are as yet
unknown.

Of course, when studying developmental phenomena, more com-
plex problems are met with. At present such phenomena as regen-
eration, budding, and cell migration have none of the convenient
environmental chemical "handles" (comparable to glutathione and
pCOo) which have so often provided the means of attacking a
problem. Yet certainly many of the environmental factors aflFecting
the feeding reflex also influence developmental phenomena. For
example. Hydra grown in a culture solution low in sodium have
smooth short tentacles and few nematocysts. At even lower sodium
concentrations the ectoderm thickens, developmental abnormalities
occur, and often cellular areas begin to disintegrate. These abnor-
malities never occur in a medium of the proper sodium content ( 11 ).

Research with a whole animal challenges the quantitative biol-
ogist. When he treats hydra with the same precision that he treats
an in vitro system, he will find that much of the mystery surround-
ing the animal disappears and that the excitement of a new under-
standing beckons.

Now let us consider the activation of the feeding reflex in
Hydra littoralis by the tripeptide reduced glutathione. We owe

HOWARD M. LENHOFF 205

the discovery of this phenomenon to two independent studies : one,
by Helen Park, who, while studying the effects of radiation on
Hydra, observed that the anti-radiation compound reduced gluta-
thione caused the Hydras mouth to open (20) ; the other by Loomis,
who, in a systematic search, identified reduced glutathione as the
substance present in crustacean extracts that activated the feeding
reflex in Hydra ( 17 ) .

The significant aspects of this discovery are many. From an
evolutionary viewpoint, data on the distribution of the glutathione-
activated response has been used to deduce the sequence in geo-
logical time that the feeding mechanisms of some coelenterates
evolved (6, 15, 17). On the whole animal level, the feeding re-
sponse is an example of an elaborate behavioral pattern controlled
by a single environmental compound. At the cellular level, the glu-
tathione-activated feeding reflex is a clear example of chemorecep-
tion specific for only one molecule.

This morning I would like to dwell on a fundamental subcellular
aspect: the mechanism by which glutathione combines with and
activates the glutathione-receptor.

DESCRIPTION OF THE NORMAL FEEDING REFLEX

All measurements are based on Hydra's characteristic feed-
ing movements, described earlier by Ewer ( 4 ) and Loomis ( 17 ) .
The drawings in Figure 1 illustrate each of these steps. A Hydra
in the absence of the glutathione has its mouth closed, and its tenta-
cles outstretched and relatively motionless. After the addition of
glutathione, the tentacles begin to writhe and sweep inwards to-
ward the longitudinal axis of the animal ( Fig. 1 A ) . Next, the tenta-
cles bend toward the mouth, and the mouth opens (Fig. IB). Shown
in this composite drawing (Fig. IB) are the various positions that a
tentacle takes before contracting. These movements, culminating in
mouth opening, usually all take place within half a minute. Figure
IC shows how a Hydra looks during the greater portion of the feed-
ing reflex, its mouth open wide and the tentacles in various phases
of contraction. Frequently, the tips of the tentacles are observed
within the Hydra's mouth, as shown in Figures IB and IC.

206

THE BIOLOGY OF HYDRA : 1961

A B C

Fig. 1. Stages of the feeding reflex (see text) (From Ref. 8).

A QUANTITATIVE ASSAY

Requisite for quantitative studies of any biological phenomenon
are accurate and reliable measurements. Therefore, special empha-
sis is placed on the assay procedure which has as its basis the visual
measurement of the mechanical process of mouth opening.

Meaningful measurements of the feeding reflex require Hydra
that respond to glutathione in a quantitatively reproducible manner.
Large numbers of such experimental animals were obtained by

HOWARD M. LENHOFF 207

starving for one or two days mass cultures of Hydra Uttoralis (18)
that had been reproducing asexually in a sokition consisting of 10~^
M CaCL and 10~^ M NaHCO;; in deionized water. Special care was
taken to remove most of the organic waste products from the cul-
tures twice daily (18). The animals in each tray were not allowed
to reach a density of over two or three thousand hydranths per 1500
ml. of culture solution.

The assay procedure used in most of these experiments was as
follows: Five starved Hydra obtained from the mass cultures were
rinsed three times in 30 ml. portions of a solution lacking gluta-
thione and consisting of 10"-^ M CaCl,, 10"^ A/ NaCl, and
10~^ M histidine chloride buffer, pH 6.2. The fixe Hydra were then
transferred in one drop of the solution into 2 ml. of the same solu-
tion containing glutathione (Sigma, St. Louis, Mo.). (Reduced
glutathione is not readily oxidizable at pH 6.2.) This glutathione
solution was in the spherical concavity (36 mm. diam. x 5 mm. deep)
of a Maximov tissue culture slide. The Hydra were immediately
observed through a binocular dissecting microscope set at a magni-
fication of 19.5. The time intervals between the moment the Hydra
were placed in the glutathione solution and the initial and final
(ti and tf) times that the mouth of each animal was open were
recorded. The magnitude of the feeding reflex is expressed as the
average time (tf-ti) during which the mouths of the Hydra remained
open in response to glutathione.

In Table 1 are shown the results of four different experiments
(a-d) which were carried out in excess glutathione. In these experi-
ments each Hydra opened its mouth within 0.4 to 1.0 minutes (ti)
after being placed in the glutathione solution. Under optimal con-
ditions, the variations observed in the opening time ti were small
when compared to tf, and did not significantly alter the o\'erall
time during which the mouth was open (tf-ti).

The closing time (tf) for the individual Hydra in each experi-
ment (Table 1, expts. a through d) was about 35 minutes. Because
the standard deviations were small in comparison to the total length
of the response, they were not routinely calculated.

At sub-optimal concentrations of glutathione (Table 1, expt. e),
or in the presence of a compound known to compete with gluta-

208 THE BIOLOGY OF HYDRA : 1961

thione for the glutathione-receptor (13) (Table 1, expt. f), some
Hydra took as long as 6 niiniites to open their mouths, while others
did not carry out the feeding reflex at all. In these cases, the stand-
ard deviation is large relative to tr-ti. Data of this type are similarly
expressed as the average time (tf-ti) during which the mouths of
the five Hydra tested remained open regardless of the number that
responded positively.

TABLE 1
Method of expressing the duration of the feeding reflex

Glutathione t^-tj ( min. )

Expt. concentration tj(min. ) tf(min.) Mean rt S.D.

(a)

10--^ M

0.43, 0.46, 0.60,
0.78, 1..33

33.00, 35.36, 38.08,
39.71, 41.00

36.71 ± 2.95

(b)

10 = M

0.50, 0.53, 0.71,
0.88, 0.91

32.80,-33.16,36.25,
36.43, .38. 11

34.64 ± 2.10

(c)

7.5 X 10-« M

0.43, 0.46, 0.58,
0.68, 0.96

28.21, 36.50, 36.50,
38.30, 43.45

.35.97 ± 5..30

(d)

5 X 10-* M

0.48,0.50,0.61,
0.78, 1.05

26.88, 35.25, 37.41,
42.00, 43.41

36.31 ± 6.36

(e)

5 X 10-7 M

0.68, 2.33, 2.63,
4.75, 00

5.08, 16.25, 22.50,
25.60,-.

11.81 ± 9.29

(f)

2 X 10-6 M

2.45, 3.75, 6.00,

6.41, 13.45, 22.91,

6.11 ±7.23

(+1C

|-^ M glutamine)

00, 00

— , — .

Hydra starved for two days were used in all experiments.
Data from reference 8.

The values for tr-ti at excess glutathione concentrations are
usually within the range of 25 or 35 minutes, depending upon
whether the Hydra were starved one or two days preceding the
experiment. This fact should be borne in mind when comparing
data from different sets of experiments. ( It now also is known that
small changes in temperature influence tf-ti significantly as shown
in Table 7. )

These data, in addition to providing a basis for the assay, give
insight into central problems concerning the mechanism by which
glutathione elicits the feeding reflex. The values given for ti must
include the time required for at least two major processes to occur:
( a ) the union of glutathione with its receptor, which in experiments
a-d is probably rapid (i.e., within a few seconds), and (b) all

HOWARD M. LENHOFF 209

of the subsequent events leading to mouth opening. The values for
ti (0.4 to 1.0 minutes) may represent, for the most part, the latter
events.

Large values of ti (those greater than 2.0 minutes) indicate that
the experimental conditions for the feeding reflex are not optimal.
For example, it takes longer for the mouth to open at low gluta-
thione concentrations (Table 1, expt. e) or in the presence of a
competitive inhibitor (Table 1, expt. f) than at excess glutathione
concentrations under optimum conditions. Similarly, cellular poi-
sons, such as N-ethyl maleimide or heavy metals, also cause an
increase in ti (9). Further, Hydra in distilled water take longer to
respond than do Hydra in distilled water containing added calcium
(10). In the cases mentioned here, it would appear that the large
values of t. result from the interference with the activation of a
sufficient number of functional receptor sites needed to elicit an
optimally rapid response. Another cause of a delay in mouth opening
might stem from interference with some of the cellular events
initiated by the combination of glutathione with its receptor.

At sub-optimal concentrations of glutathione (Table 1, expt. e,
and Fig. 3 at concentrations less than 5 ■ 10"*' M glutathione) the
tf-ti values were small in comparison to those obtained at higher
glutathione concentrations. These results show that graded responses
can occur when conditions are not optimum. In addition, it is gen-
erally observed that the larger the value of ti, the smaller the value

of tf-ti.

EFFECT OF GLUTAMIC ACID AND
GLUTATHIONE ANALOGS

Using glutathione analogs, we have undertaken a study of the
size and configuration of the glutathione molecule necessary for
activation of the response. The results, summarized in Table 2,
show that the glutathione-receptor has a most unusual specificity
compared to proteins which react with glutathione ( 13 ) . The recep-
tor (a) is not dependent upon the sulfhydryl moiety of glutathione
for activation, (b) has a high order of specificity for the structure
of the tripeptide "backbone" of glutathione, and (c) is inhibited
by glutamic acid.

210

THE BIOLOGY OF HYDRA : 1961

TABLE 2
Activators and inhibitors of the feeding reflex

CH.

O.C-CH-CH.-CH.-CO-

NH-CH-CO-

NH-CH.-CO-2

lutamyl -

alanyl

- jjlycine

Activators

Inhibitors

( tripeptide )

( others )

R = -H
R = -CH.
R = -SH
R = -S-CH .,

R = -SO.H

R = -SO:;H

R = -S-COC
R = -S(N-et
R = _S-SG

H:,

lylsuccininiido)
-CH.CO-

glutamic acid

glutamine

cysteinylglycine

R = SH

and

A =-0,C-CH

1
+NHo

In confirmation of Cliffe and Waley ( 3 ) , we were able to extend
their striking resnlts demonstrating that the sulfhydryl group is not
necessary for the action of gkitathione on Hydra and that this group
can be altered within certain limits. These workers obtained a posi-
tive response in Ilijdm exposed to the lens tripeptide ophthalmic
acid (y-glutamyl-«-amino-/]-butyryl-glycine). This tripeptide, as
well as nor-ophthalmic acid, activates the feeding reflex in Hydra.
Both peptides are identical to glutathione except that they have
respectively a methyl or a hydrogen atom instead of the sulfhydryl.
We further show that the S-methyl analog of glutathione also acti-
vates the feeding response ( 13 ) .

On the other hand, substitution of large groups for the sulf-
hydryl moiety leads to analogs which do not have the right
configuration to activate a response. Rather, such analogs
(y-L-glutamyl-L-sulphi-alanylglycine, y-L-glutamyl-L-sulpho-alanyl-

HOWARD M. LENHOFF 211

glycine, S-acetyl glutathione, S-succinimido glutathione, and
oxidized glutathione ) when at high concentrations inhibit the action
of glutathione (3, 13). These inhibitions are overcome by increasing
the glutathione concentration. Thus, those analogs which retain the
tripeptide backbone of glutathione act as competitive inhibitors in
the activation of the feeding reflex.

Another tripeptide which acts as a competitive inhibitor is
asparthione (/?-aspartylcysteinylglycine) (13). This compound is
nearly identical to reduced glutathione except that it lacks one
methylene group, having aspartic acid substituted for glutamic
acid. Loomis first showed that asparthione fails to activate the feed-
ing reflex ( 17 ) . These characteristics of asparthione point out the
importance of the y-glutamyl moiety of the tripeptide for the acti-
vation process, as well as providing additional proof that the pres-
ence of a sulfhydryl group on a tripeptide similar to glutathione
is not sufficient for activity. Contrastingly, glyoxylase, another glu-
tathione-requiring system, functions with asparthione (1).

Further evidence that the y-glutamyl moiety is of special impor-
tance in the active structure of glutathione is the action of both
glutamic acid and glutamine as competitive inhibitors of gluta-
thione, while neither cysteine nor glycine have this efi^ect ( 13 ) .
The importance of the a-amino group of the glutamyl moiety is
emphasized by the failure of a-keto glutaric acid and of glutaric
acid to inhibit. Also, as might l)e anticipated, neither aspartic acid
nor asparagine were inhibitory (13).

These data indicate that the receptor has a high affinity for the
y-glutamyl group, that the sulfhydryl group is important only
in that it conforms to certain size limitations, and that the glycine
is needed to complete the fit of the tripeptide into the receptor
(Loomis has shown that y-glutamylcysteine does not activate a
response. Ref. 17). As more analogs become available, we hope to
determine the exact structural requirements for the stimulatory
activity of glutathione. In addition, it should be possible by com-
paring the Ki's of the different inhibitors to determine the relative
affinities of the receptor for the different parts of the glutathione
molecule.

No other system known to require glutathione has such exacting
requirements for the peptide backbone of glutathione. Regardless

212 THE BIOLOGY OF HYDRA : 1961

of this remarkable specificity and of the ample glutathione in the
fluids emitted from Hydra s captured prey in nature, the possibility
remained that some unknown trace factors present in these fluids
are the natural activators of the feeding reflex.

To examine this possibility, the following series of experiments
were carried out ( Table 3 ) : a diluted aqueous extract ( 30;iig. )
from homogenized Arfemia elicited a 37 minute feeding response
in Hydra (expt. 1). A similar extract containing 10~^ M added
glutamic acid gave only a 7 minute response (expt. 2). The glu-
tamic acid was presumably competitively inhibiting the glutathione
in the Artcmia extract because, if in addition to 10~^ M glutamic
acid, 10~"' M glutathione, was also included, then the inhibition
was reversed ( expt. 3 ) . The inhibition was also reversed by increas-
ing the amount of shrimp extract (expt. 4). Further evidence that
the glutamic acid was acting competitively and was not irreversibly

TABLE 3

The inhibition by glutamic acid of the feeding reflex induced by Artemia
extracts, and the reversal of that inhibition by glutathione

Expt. Test Solution tj-t;

( mill. )

1. 30 p,g. Artemia extract. 37.3

2. 30 fig. Artemia extract and

10^ M glutamic acid. 7.4

3. 30 /ig. Artemia extract,
10~* M glutamic acid, and

10"^ M reduced glutathione. 40.6

4. 140 ng. Artemia extract and

10-' M glutamic acid. 26.5

5. Hydra from expt. 2 in

10"" M reduced glutathione. 29.1

6. 10-" M reduced glutathione 42.1

Hydra starved for two days were used in all experiments.

HOWARD M. LENHOFF 213

poisoning the Hydra was shown in experiment 5 where the inhibited
animals from experiment 2 were washed and then immediately
placed in a fresh glutathione solution; these inhibited Hydra
responded again for an additional 29 minutes. These experiments
leave little doubt that reduced glutathione emitted from the prey
is the major natural substance activating the feeding reflex in Hydra
littoralis.

EFFECT OF ENVIRONMENTAL CATIONS AND ANIONS

As emphasized earlier, the Hydra's external aqueous environ-
ment takes on a special importance when studying the feeding
reflex. This fluid serves a dual role: it conveys glutathione from the
prey to Hydra, and it bathes both the receptor and the ecto-
dermal effector cells which are involved in part of the contractile
processes of the feeding reflex. Therefore, before directly investigat-
ing the mechanism by which glutathione activates the response, it is
necessary to consider the influence on the feeding reflex of the ions
in the media bathing the animals. Knowledge of the effects of these
ions might be useful in gaining insight into the mechanisms involved

TABLE 4
Ionic requirements for the activation of the feeding reflex

Cations

Anions

Expt.

Text Solution

tf-ti

Expt.

Test Solution

tf-tj

(min.)

10-3 M

(min.)

10-^ M EDTA

l.a.

CaCL.

28.5

10-^ M EDTA and
10-=' M CaCLo

28.5

2.a.

CaBr.

23.0

10-^ M EDTA and

3.a.

CaL.

9.2

10-=' M SrCL

6.8

4.a.

Ca(NO.).

8.3

Hydra starved for one day used in all experiments.

All ions were dissolved in a solution of 10-* M NaHCOs

Data from reference 10.

214

THE BIOLOGY OF HYDRA : 1961

in the feeding reflex in addition to defining the experimental limits
within which the ionic composition can be varied.

Summarized in Table 4 are data concerning the ionic require-
ments for the feeding reflex (10). Hydra placed in a 10~^ M
solution of the chelating agent ethylenediamine tetraacetic acid
(EDTA) lost their ability to respond to glutathione (expt. 1). Since
EDTA is known to chelate calcium ion, one of the two environmen-
tal cations required for the growth of Hydra, 10~'' M CaCL was
added to this same solution, and the Hydra responded normally
(expt. 2). No other cation would replace calcium to any degree in
reversing the inhibitory action of EDTA except strontium ( expt. 3 ) .
Since this metal behaves chemically like calcium, experiment 3
strengthens the evidence that calcium is required to effect the
feeding reflex.

Further evidence for the calcium requirement was obtained

0.4 r-

--0.2 -

[CO-H+]

Molarity x 10 ^)

Fig. 2. The inhibition of the feeding reflex by magnesium ions, and its
reversal by calcium ions.

HOWARD M. LENHOFF 215

using magnesium ions, an ion known to compete with calcium in
many biological systems. To show the competitive nature of the
magnesium inhibition, our data is expressed in a fashion analogous
to the Lineweaver-Burke plot. Here (Fig. 2) we plot the reciprocal
of the duration of the feeding reflex against the reciprocal of the
calcium concentration. These experiments were carried out in the
absence of magnesium, or in 10~^ M MgCL, or in 10~^ M MgCL.
The data show that the higher the concentration of magnesium,
the greater is the inhibition. Furthermore, as the calcium concen-
tration is increased, the magnesium inhibition is completely re-
versed. These experiments leave little doubt that magnesium is
interfering with the normal function of calcium ions in the feeding
reflex. Sodium ions also exhibit similar competitive inhibitory efl^ects
(9). However, for a comparable inhibition higher concentrations of
sodium ions are necessary.

At present there is little evidence as to whether the calcium
functions at the glutathione-receptor, or in the efl^ector system. (It
appears that the trypsin activation of the feeding reflex, which will
be described later, also requires environmental calcium, thus favor-
ing the involvement of calcium in the effector system.)

Anions were also found to influence the feeding reflex (Table
4 ) . Holding the calcium content constant, the order of effectiveness
of the anions in increasing the duration of the feeding reflex was:
CI > Br > I = NO:, (10). The relationship of this order to the lyo-
tropic series suggests that these ions influence the state of some
proteins involved in the feeding reflex.

From a practical viewpoint, these results point out the necessity
of controlling precisely the ionic environment for the quantitative
study of the feeding complex.

COMBINATION OF GLUTATHIONE WITH
THE RECEPTOR

Most of the data just described concerns environmental chem-
istry. Now we have to ask questions about the first physiological
event of the feeding reflex, the combination of glutathione with
the receptor.

216 THE BIOLOGY OF HYDRA : 1961

TABLE 5
Time required for mouths to close on removal of glutathione

Time in Glutathione

Time to Close

( min. )

5.0

0.94

10.0

0.92

12.5

0.99

15.0

0.76

20.0

0.72

22.5

0.44

25.0

0.41

All experiments were carried out using Hydra starved for one day, and at excess
glutathione ( 10"^ M).

Data from reference 8.

The following simple experiment demonstrated that glutathione
did not act as a "trigger," if a triggered response is defined as one
that continues after the initial stimulus is removed. Groups of Hydra
were incubated in 10"'' M glutathione for periods varying from 5 to
25 minutes (Table 5). At the end of each incubation period, the
animals in one drop of glutathione solution, were placed in 30 ml.
of a solution of the same composition but lacking glutathione. In
all cases the mouths closed in less than one minute (Table 5). The
results indicate that glutathione had to be constantly present during
the total time of the feeding reflex in order for the response to con-
tinue. In addition, since the mouths close repidly on removal of
glutathione it is concluded that the equilibrium between glutathione
and the receptor is rapidly attained.

This observation that the continued presence of glutathione is
required for the activation of the feeding reflex allows us to formu-
late a hypothesis on how glutathione activates the receptor. We
visualize the receptor as an inactive protein on the siuface of
certain Hydra cells. When that protein combines with glutathione,
its tertiary structure is altered, rendering the receptor protein physi-
ologically active. The active protein is then capable of either initi-
ating, or allowing to go on to completion, the events involved in
the receptor-eflFector system.

These data also indicate that the longer Hydra were exposed
to the glutathione, the sooner the mouths closed when glutathione

HOWARD M. LENHOFF 217

was removed. The time that it took for the mouths to close prob-
ably represent the time required both for the dissociation of the
glutathione and for the cessation of the cellular events involved
in the receptor-effector system. The observations that mouth closure
was more rapid the longer Hydra were exposed to glutathione may
imply that bound metabolic intermediates or cofactors, postulated
to be released by and to take part in this system (12), become
depleted as the feeding reHex nears completion.

When considering the quantitative aspects of the union of gluta-
thione with the receptor, we found that the data were more mean-
ingful if they were treated according to concepts borrowed from
enzymology. Therefore, we investigated the effect of glutathione
concentration on the "activity" of the receptor-effector system, the
"activity" in this case being expressed as the duration of the feeding
reflex (Fig. 3). For each concentration of glutathione, duplicate
groups of five animals were used. In experiments employing Hydra
starved for two days (solid curve), a maximum response was ob-
served at concentrations of 5 X 10 ""^ M and greater. No further
increase in the duration of the feeding reflex occurred at higher
glutathione concentrations. At lower glutathione concentrations,
the duration of the feeding reflex increased in nearly direct propor-
tion to the amount of glutathione added. However, at these smaller
values there was greater variation in the response of the individual
Hydra, some not responding at all, as indicated by the symbols
used in Figure 3. The similarity of this plot (Fig. 3) to the Lang-
muir adsorption isotherm, and to a curve illustrating the saturation
of an enzyme by its substrate is apparent. Accordingly, the results
(Fig. 3) are interpreted as indicating that at glutathione concen-
trations greater than 5 X 10"** M all of the glutathione-receptors are
saturated. In these experiments we have not been able to demon-
strate that the glutathione is metabolized in a manner analogous
to the metabolism of a substrate by its enzyme. But rather it appears
as if the glutathione continues to activate all of the receptor-
effector systems until the response ceases. At subsaturation levels
of glutathione, the animal does not respond to its fullest capacity
(see also Table 1, expt. e).

Another useful concept, analogous to the Michaelis constant, or
K^r, used in enzymology, is the concentration of glutathione eliciting

218 THE BIOLOGY OF HYDRA : 1961

a half-maximum response. For the g\utathione-H ydm system this
value, ca. 10"^ M, probably closely approximates a true dissociation
constant because of the apparent absence of glutathione metabolic
products. A rough mass law treatment using the method of Scatch-
ard (21) indicates that this constant can be measured within a
factor of 2. The significance of this constant is threefold: First, its
smallness indicates that the receptor has a high affinity for gluta-
thione. Second the value of 10~^ M is within the physiologically
active range expected to occur under natural conditions of feeding.
And, third, this number provides a means of characterizing the
receptor; that is, the glutathione receptor can be said to have a
constant of 10~^ M. This constant has been found to be a charac-
teristic of the receptor and to be nearly the same no matter what
the nutritional state of the Hydra. For example, Figure 3 demon-
strates that Hydra starved for two days respond to higher concen-
trations of glutathione for a greater period of time than do Hydra
starved for one day (lower curve). Nonetheless, the concentration
of glutathione eliciting a half-maximal response on both sets of
Hydra was 10~^ M.

The difference in the maximum response observed in Hydra
starved one or two days ( Fig. 3 ) become understandable if another
comparison is made with enzyme systems. Just as the maximum
activity of an enzyme reaction is dependent on the quantity of
enzyme present and is not a specific property of the enzyme, in a
similar manner the duration of the reflex at concentrations eliciting
the maximum response is dependent upon the quantity of com-
pleted^ receptor-effector systems of the Hydra. The maximum
response is not an intrinsic property of the receptor or of Hydra
as is the Km. Thus, Hydra starved for one day are interpreted to
have fewer completed receptor-effector systems than Hydra starved
for two days.

As emphasized in the above comparison, just as the total enzyme
activity at saturating concentrations of substrate is proportional to
the amount of enzyme, so the total maximum response of Hydra to

lA completed receptor-effector system is defined as one containing all of the com-
ponents necessary for it to function when in combination with glutathione. When
all the receptor-effector systems are completed, the Hydra is capable of carrying
out a maximum response.

HOWARD M. LENHOFF

219

HYDRA STARVED FOR TWO DAYS

GLUTATHIONE CONCENTRATION (Molorify x 10")

Fig. 3. Effect of glutathione concentration on the duration of the feeding
reflex. Each point represents the mean for five Hydra. The type of symbol
used indicates the number of Hydra in the group of five responding to
glutathione: i.e. o, five; •, four; [H, three; A/ fwo; and A/ one. (From
reference 8).

excess glutathione is proportional to the number of active receptor-
effector systems in each Hydra. Thus, in order to get comparable
results, it is imperative in experiments using excess glutathione
concentrations (10^' M) that each Hydra possess approximately
the same number of such systems. Since it is impossible to know
beforehand the number of completed receptor-effector systems per
Hydra, the only criterion for obtaining quantitatively reproducible
results is to select Hydra reared under nearly identical laboratory
conditions. We repeatedly find that the standard deviation of the
response of Hydra to excess glutathione is lov^^ if these animals
come from the same mass culture (Table 1, expts. a-d). Therefore,
one should not compare experiments employing Hydra taken from
different mass cultures. Variation might result either from differ-

220

THE BIOLOGY OF HYDRA : 1961

ences in the following factors: the time elapsed since the previous
exposure to glutathione, the ratios of environmental cations or
anions, the temperature of the experiment, or to some presently
unidentified factors.

EVIDENCE FOR AN INTRINSIC LIMIT
TO THE RESPONSE

The data in Table 1 and Figure 3 show that the feeding reflex
is limited to 25-35 minutes, depending upon the conditions of the
experiments. In order to determine whether this mouth closure
resulted from some intrinsic change within the Hydra, or from the
oxidation or alteration of glutathione in the culture solution the
following experiment was performed: A group of 5 Hydra were
exposed to 2 ml. of 10~^ M glutathione until their mouths closed
(Table 6). The same glutathione solution was then transferred to

Response of

TABLE 6

different groups of Hydra exposed to the some solution of
excitatory compound used three times

DURATION OF FEEDING
tf-tj (mill.)

REFLEX

Group of Hydra

Glutathione Ophthalmic Acid
10-= M lO-fi M

S-Methyl

Glutatliione

10-= M

1
2
3

27.1 27.6
19.8 23.1
28.1 24.0

29.5
26.2
21.7

AU Hydra were starved for one day.
Data from reference 8.

another group of 5 Hydra; this latter group of Hydra opened their
mouths for 27 minutes, indicating that sufficient glutathione re-
mained to elicit a near-maximum response. This transfer process
was repeated, and again the Hijdra responded positively, although
for a somewhat shorter time. Using the p-mercuribenzoate proce-

HOWARD M. LENHOFF 221

dure of Boyer (2), parallel experiments were run in which the
respective solutions were assayed for sulfhydryl groups after the
Hydra had closed their mouths. No perceptible decrease in the
sulfhydryl content of the solution occurred.

Similar experiments were carried out using the glutathione ana-
logs ophthalmic acid and S-methyl glutathione, compounds that
activate the feeding reflex and are not auto-oxidizable. As shown
in Table 6, these analogs like glutathione, retain much of their activ-
ity after several exposures to Hydra. It can be concluded from all
the experiments summarized in Table 6 that the feeding reflex nor-
mally ends as a result of some other cause than the oxidation, disap-
pearance, or alteration of the glutathione molecule; also it does not
end because of the accumulation of inhibitors in the culture solution.

Further examination of Table 6, however, does indicate some
shortening of tf after using the same glutathione solution on three
successive groups of Hydra. Thus, it appears that either the gluta-
thione concentration was in some manner slightly lowered, or that
some inhibitory factor gradually accumulated in the environment.

It is not necessary to assume that the glutathione or glutathione
analogs are altered or destroyed when combining with the receptor.
There are known instances in which a biological response is initi-
ated by a molecule (non-coenzymic in function) combining with
a specific site without being metabolized. For example, thiogalacto-
side induces the adaptive fonnation of the enzyme /?-galactosidase
without being hydrolyzed ( 19 ) .

Thus, from the data in Table 6, we might postulate as one result
of glutathione activation, the consumption of some substance in
the receptor-effector system, the concentration of which limits the
duration of the feeding reflex to 25-35 minutes. If this postulate is
true, then one might expect that after the Hydra have carried out
a maximum response, there will be a period during which they
give no further response to a fresh solution of glutathione. Secondly,
there will be another period in which they regain their ability to
respond maximally. This proved to be the case as shown in Figure
4. In this experiment large numbers of Hydra were exposed to gluta-
thione for forty minutes. The animals were then washed with and
placed into the glutathione-free culture solution, and, at intervals,
exposed to a fresh solution of glutathione. The results show that

222 THE BIOLOGY OF HYDRA : 1961

40 I-

10 20 30 40

HOURS AFTER INITIAL EXPOSURE TO 6SH

Fig. 4. Time for recovery of the ability to respond to glutathione (see
text).

during the first hour, the animals give Httle if any response. By the
tenth hour, however, the Hydra had regained their abihty to respond
for about 15 minutes, and after one day, responded maximally.
Extending the interval between exposures to o\'er 70 hours did not
result in any further increase in the length of their response to fresh
glutathione.

This lag and gradual resumption in the ability of Hydra to
respond to a fresh stimulus of glutathione is interpreted to signify
the period for the resynthesis of some substance, called "X," which
we postulate to be limiting in the receptor-effector system. This
view places a greater emphasis on the state of the receptor-effector
system than on the physiology of the whole animal.

EFFECTS OF TEMPERATURE

The effects of temperature on the feeding response were studied
primarily to provide more evidence concerning limiting substance X

HOWARD M. LENHOFF

223

and information concerning its role in the execution of the response.
These experiments are still in the notebook stage and will be sum-
marized here only in order to show you some of the directions our
research is taking.

If the reactions of the feeding reflex (Fig. 5) are depicted as in-
x'olving the conversion of limiting substance X to Y, then one might
expect two major results of lowering the temperature. First, a small
lowering in temperature should lower the rate of all the thermo-
chemical reactions. However, by slowing down the reaction con-
verting the X to Y we should therefore slow down the rate at which
the supply of X is depleted, and thus increase the length of time
that the mouth remains opened. This proved to be the case as shown
in Table 7 where, as the temperature approaches 15 \ the Hi/dm
respond for nearly 100 minutes.

TABLE 7
Effect of temperature on the duration of the feeding reflex

Temperature

tf-ti

Temperature

tf-ti

( min. )

(min.)

6.2°

5.9

18.1°

86.9

8.9°

36.4

18.6°

55.0

10.3°

59.8

19.7°

59.2

12.5°

70.7

20.6°

.55.0

14.5°

60.0

21.9°

35.1

15.4°

88.6

24.1°

29.4

16.3°

99.7

25.3°

21.5

27.7°

19.7

All Hydra were starved for two da) s.

As a second efl^ect of lowering temperature, the limiting reaction
may go so slowly that the optimum (threshold) conditions neces-
sary for the feeding reflex are not maintained. Thus, when the
Hydra are held below 15° they are observed to open their mouths
for a few minutes, then close, open, etc. until they finally stop
responding. The total duration of the responses below 15 becomes
progressively less until the Hydra barely respond (Table 7). In fact,
when the temperature is lowered from 20" to 5", the mouth takes

224 THE BIOLOGY OF HYDRA : 1961

longer to open (Table 8). These results (Table 8) are interpreted
to mean that as the temperature is lowered, it takes longer for the
completion of all the reactions (including the limiting one) leading
to mouth opening.-

TABLE 8
Effect of temperature on time of mouth opening

Temperature 1 /t,

(min. )

5.2°

0.10

7.9°

0.35

9.5°

0.62

13.0°

1.11

16.3°

1.72

18.5°

1.56

20.1°

2.56

All Hydra were starved for two days.
Each value is the mean for 25 animals.

ACTIVATION BY PROTEOLYTIC ENZYMES

Recently we have been carrying out some experiments in acti-
vating the feeding response in the absence of added glutathione by
using certain proteolytic enzymes. Although still in the preliminary
stage, these experiments may help illucidate the sequence of events
taking place in the receptor-effector system, and thus are of sufficient
interest for some of them to be reported here.

We have previously shown that papain, ficin, and trypsin acti-
vate a feeding response in Hydra (12). This proteolytic activation
was shown not to be the result of any toxic action of the enzymes
for the Hydra were intact and alive after one day's exposure to the
proteases. Dialyzed ficin, like papain (Table 9), did not activate
a response unless cysteine was added to render the enzyme active.
The boiled enzymes could not be activated by cysteine. The action

-At temperatures below 13° Hydra vary greatly in their response, some animals not
responding at all. Therefore, the data are expressed as 1/tj rather than as tj because
in cases where there was no response, the t; values would range to infinity.

HOWARD M. LENHOFF 225

of trypsin was inhibited by trypsin inhibitor (Table 9). Thus, the
response seems to be a result of the proteolytic activities of these
enzymes. Of twenty other purified proteins, only chymotrypsin gave
a significant (8 min.) response (12). It does not seem likely that
the proteases are acting by releasing reduced glutathione from
Hydra because y-glutamyl linkages are rare in proteins, and because
furthermore glutamic acid, a specific inhibitor for glutathione (13),
does not inhibit the action of trypsin (9) .

The possible effects of proteases on a whole animal are so numer-
ous that it would be difficult at this time to single out any one
action that would explain their effect on Hydra. Nevertheless, the
important fact remains that proteases do activate a response, and
thus a study of their effects might help in arriving at an understand-
ing of the actual mechanism. For example, trypsin can activate
only an 18-minute response; if glutathione, however, is added to
the same Hydra, they respond an additional 17-18 minutes. In con-
trast, after a 35 minute response initiated by glutathione, the addi-
tion of trypsin has no effect. A mixture of excess glutathione and
excess trypsin, interestingly enough, elicits a response equal only
to that initiated by glutathione alone. Thus, these preliminary exper-
iments indicate that the protease probably activates a series of
events common to those activated by glutathione and involving
the consumption of limiting substance X (9). Therefore, in Figure
5, the arrow indicating the site of action of the protease is drawn

TABLE 9
Activation of feeding reflex by proteases

Expt. Test Solution t^-tj

(min.)

La. 20 /ig./ml. papain 0.1

b. 20 fig./ml. papain -|- 10'3 M cysteine 19.8

c. 20 /ig./ml. papain -|- 10'3 M cysteine, boiled 5 min. at 100° 0
2.a. 0.1 mg./ml. trypsin 17.8

b. 0.1 mg./ml. trypsin -(- 0.1 mg./ml. trypsin inhibitor 0

All Hydra were starved for two days.
Data from reference 12.

226 THE BIOLOGY OF HYDRA : 1961

somewhere between the receptor and before the reaction involving
the consumption of X.

The activation by proteases has also been useful in determining
the relative site at which calcium functions. Since the presence of
environmental calcium ion is required for the activation of both
glutathione (10) and proteases (9), we feel that calcium plays a
role in the effector system rather than in the combination of gluta-
thione with the receptor.

A recent development which places added importance upon the
activation of the feeding reflex by f)roteases is the discovery by
Fulton (6) that proteases also activate the feeding reflex in Cordy-
lophora. His results are striking in that he has also shown that
Cordylophora do not carry out the feeding reflex in response to the
peptide reduced glutathione, but rather to the single imino acid pro-
line (5). Thus, although Hydra and Cordylophora have different
specific excitatory compounds, the feeding reflex in both animals can
be activated by proteases. In addition, Physalia gastroozoids, which
normally respond to glutathione ( 15 ) , also are activated by i)ro-
teases ( 12 ) . All of these results suggest that the protease is acting
on some step which is common to the feeding reflex of all these
organisms irrespective of the excitatory compound involved.

SUMMARY AND CONCLUSIONS

With the aid of the simplified scheme shown in Figure 5, I
would like to summarize the present state of knowledge concerning
the mechanism by which glutathione combines with and activates
the glutathione-receptor of Hydra to elicit the feeding reflex. The
activity of the glutathione resides in the size and configuration of the
7-glutamylalanylglycine backbone of the tripeptide, and not in the
reducing properties of the molecule ( 3, 13, 17 ) . Concentrations of
glutathione greater than 5 X 10 ~"' M activate all of the receptor-
effector systems (Fig. 3), which are probably localized in the area
immediately around the mouth and on the tentacles (8). The con-
centration of glutathione eliciting a half maximum response is 10~*^
M (Fig. 3). In order for a response to take place, the glutathione
must be constantly present at the receptor site (Table 5). The associ-

HOWARD M. LENHOFF 227

ation of glutathione with the receptor is rapidly attained ( Table 5 ) ;
the affinity of the receptor for glutathione is high (Fig. 3). After
glutathione combines with the receptor, it takes about 0.5 minutes
for all the events necessary for mouth opening to occur ( Table 1 ) .
Once the reflex begins, it will continue for 25 to 35 minutes (Tables
1, 3, 4 and 6; Figs. 3 and 4). The response does not stop because of
any alteration in the glutathione molecule (Table 6), but rather
because of some inherent property of Hydra. The duration of the
response is probably directly related to the conversion of some lim-
iting substance X to its product Y (Tables 1, 3, 4 and 6). Lowered
temperatures increase the duration of the feeding reflex, probably
by decreasing the rate at which the supply of X is exhausted ( Table
7 ) . It takes about 24 hours for X to be resynthesized either from Y
or anew (Fig. 4). The response can be stimulated in the absence
of glutathione by certain proteases (12). The protease probably
acts before the step involving the consumption of X. Furthermore,
the presence of small amounts of calcium ion in the medium sur-
rounding Hydra are required in order that a response may occur
(10). The calcium appears to be involved in steps occurring between
the site of activation by proteases and the effector system.

X Y

V f ^^Feeding

GSH4-Rec^[GSH-Rec] — >E,-^E„-^=^E, »E., <^j>

I ^k- Reflex

Protease

Fig. 5. Schematic outline of the glutathione receptor-effector system.
Rec represents the receptor; E,„ E,,, E,, and E,„ enzymes; X, the limiting
substance; and Y, its metabolic product.

These results are concerned with a single biological system in
which a specific excitatory compound combines with its receptor
to activate a coordinated response. Activations by an excitatory com-
pound comprise the common step in many basic biological phe-
nomena such as chemoreception and hormone action. Some of the

228 THE BIOLOGY OF HYDRA : 1961

results described here on the interaction of gkitathione with the
Hydra receptor may bear a relation to the functioning of some of
these other systems.

ACKNOWLEDGEMENTS

It is a pleasure to acknowledge the superb assistance that Mr.
John Bovaird has provided throughout this study. The criticisms
of this manuscript by Drs. J. F. Woessner, Jr., A. Phillips, E. L.
Chambers, and W. D. Dandliker are greatly appreciated.

REFERENCES

1. Behrens, O. 1941. Coenzymes for glyoxylase. }. Biol. Chein. 141: 503-508.

2. BoYER, P. D. 1954. Spectophotometric study of the reaction of protein sulfhydr\'l

groups with organic mercurials. /. Am. Cheiri. Soc. 76: 4331-4337.

3. Cliffe, E.. E., and S. G. Waley. 1958. Effect of analogues of glutathione on

the feeding reaction of hydra. Nature 182: 804-805.

4. Ewer, R. F. 1947. On the function and mode of action of the nematocysts of

Hydra. Proc. Zool. Soc. London 117: 365-376.

5. Fulton, C. 1960. The biology of a colonial hydroid. Ph.D. Thesis. The Rocke-

feller Institute.

6. Fulton, C. (in press). The growth and feeding of Cordylophora and other

hydroids. In The Lower Metozoa: Comparative Biology and Phtjlogeny,
edited by M. B. Allen. Academic Press, Inc., New York.

7. Lenhoff, H. M., 1961. Digestion of protein in Hydra as studied using radio-

autography and fractionation by differential solubilities. Exptl. Cell
Research 23: 335-353.

8. Lenhoff, H. M. (in press). Activation of the feeding reHex in Hydra littoralis.

I. Role played by reduced glutathione, and quantitative assay of the
feeding reflex. /. Gen. Physiol.

9. Lenhoff, H. M. Unpublished observations.

10. Lenhoff, H. M. and J. Boviard. 1959. Requirement of bound calcium for the

action of surface chemoreceptors. Science 130: 1474-1476.

11. Lenhoff, H. M. and J. Bo\aird. 1960. The requirement of trace amounts of

environmental sodium for the growth and development of Hydra. Exptl.
Cell Research 20: 384-394.

12. Lenhoff, H. M. and J. Bovaird. 1960. Enzymatic activation of a hormone-like

response in Hydra by proteases. Nature 187: 671-673.

13. Lenhoff, H. M. and J. Bovaird. 1961. Action of glutamic acid and glutatliione

analogues on the Hydra glutathione-receptor. Nature 189: 486-487.

14. Lenhoff, H. M. and W. F. Loomis. 1957. Environmental factors controlling

respiration in hydra. /. Exp. Zool. 134: 171-182.

15. Lenhoff, H. M. and H. A. Schneiderman. 1959. The chemical control of

feeding in the Portuguese man-of-war, Physalia Physalis L. and its bearing
on the evolution of the Cnidaria. Biol. Bull. 116: 452-460.

HOWARD M. LENHOFF 229

16. LooMis, W. F. 1954. Environmental factors controlling growth in livdra. ]. Exp.

Zool. 126: 223-234.

17. LooMis, W. F. 1955. Glutathione control of the specific feeding reactions of

h\dra. Ann. N. Y. Acad. Sci. 62: 209-228.

18. LooMis, W. F. and H. M. Lenhoff. 1956. Growth and sexual differentiation

of hydra in mass culture. /. Exp. Zool. 132: 555-574.

19. MoNOD, J. 1956. Remarks on the mechanism of enzyme induction. In Enzymes:

Units of Biological Structure and Function, edited by O. H. Gaebler.
Academic Press, Inc., New York, pp. 7-28.

20. Park, H. D. 1953. In \V. F. Loomis, reference 17, p. 211.

21. ScATCHARD, G. 1949. The attraction of proteins for small molecules and ions.

Ann. N. Y. Acad. Sci. 51: 660-672.

DISCUSSION

LANE: Would you care to speculate about the nature of the gluta-
thione-receptors, and their location?

LENHOFF: I can only guess that the receptor is a very specific
protein, probably a lipoprotein on the cell membrane. The evidence
is not too good concerning the location of the receptors on Hydra.
Experiments using isolated parts of Hydra show that some are lo-
cated on the tentacles, and others on the hypostome. We tried to
localize the receptor by radioautography using glutathione. But
the glutathione washes readily off.

SLAUTTERBACK: Aren't you inhibiting the oxidative enzymes
severely when \'0u get down to 6 degrees and thus reduce the gen-
eral motility of the animal?

LENHOFF: No doubt we are slowing down many reactions by
lowering the temperature, but the limiting reaction is the one that
we think causes this delay in mouth opening.

SLAUTTERBACK: Are these animals still moving around actively?

LENHOFF: Yes. In assaying the feeding reflex, we observe mouth
opening, tentacle waving and contraction. All these movements
seem normal as does contraction after a mechanical stimulus.

BURNETT: Maybe you could explain this preliminary experiment.
We placed hydra in a 10~^' M solution of glutathione and waited
until all of them had closed their mouths and discontinued their

230 THE BIOLOGY OF HYDRA : 1961

feeding response. At this time we offered the hydra several hun-
dred brine shrimp. The hydra readily captured, killed, and ingested
the shrimp.

LENHOFF: I can give some explanation. When a Hydra punc-
tures a shrimp, all sorts of new and unknown substances present
in the body fluids of the shrimp flow into the media. There is a
possibility that these emitted fluids contain substances which en-
hance the feeding reflex. In fact, we have some preliminary indica-
tions that phospholipids present in serum do just this. Since I think
that the initial activation takes place on the cell membrane, it is
possible that the phospholipids act there.

BURNETT: I suppose it is enhancing something already present.

LENHOFF: Yes. The point I want to emphasize is that it is very
hard to know what is happening since you do not know what is
present in the fluids coming out of the shrimp. So many factors
affect the feeding reflex, as I have shown you already.

Chandler Fulton also shows that Cordylophora respond some-
what to shrimp after they no longer respond to proline. This may
be a similar phenomenon.

GOREAU: Have you tried amino acids? I ask because we recently
noted that small amounts of methionine caused corals to extrude
mesenterial filaments. The entire colonies become covered with
tangled white masses of filaments that stayed out as long as the
methionine (2 /xglO ml) was in the medium. Extrusion of masen-
terial filaments is a typical response of some corals when feeding
in the presence of thick plankton swarms, but I have never seen
such a strong sustained reaction with other stimulants, including
clam juice, as with methionine.

LENHOFF: I haven't tried methionine, although I doubt whether
it would cause Hydra to respond. I fully agree that other com-
pounds may work on other organisms. Fulton, for example, has
shown that proline activates the feeding reflex of Cordylophora.

GOREAU: Glutathione seems to have little effect on those corals
on which it was tried. Zooplankton swarms probably secrete detec-

HOWARD M. LENHOFF 231

able amounts of all kinds of organic substances which activate
chemoreceptors to trigger the corals' feeding posture. Corals feed
any time there is plankton around. The classic story that reef corals
expand only at night is untrue. In fact, we have frequently seen
corals feeding on swarms of zooplankton in the middle of the day
irrespective of light intensity, using tentacles and extruded mesen-
terial filaments to catch and entangle their prey.

LENHOFF: It would he nice to see whether methionine analogs
will inhibit this response in corals elicited by clam juice. This would
provide strong evidence that methionine is the actixe compound
in the clam juice.

STREHLER: Langdon found that the reduced chain of insulin
is a competitive inhil:)itor of glutathione-TPN reductase. Have you
tried reduced insulin?

LENHOFF: We have not tried insulin or reduced insulin yet. But
Langdon's finding places this experiment high on our list.

Another point I find exciting is that Langdon calls insulin a
"prohormone." That is, he suggests that insulin will not work unless
it is first split, although here it is split by reduction. Thus, insulin
may represent a case of an excitatory compound being activated
by the unmasking of an essential group. We think that unmasking
phenomena (possibly proteolytic) may operate in control systems
generally.

BURNETT: Did you repeat the experiments of Balke and Steiner
showing that lactic and ascorbic acids elicited a feeding reflex?

LENHOFF: Yes. I found neither lactic nor ascorbic acid to work.
However, I still wouldn't be surprised if under certain conditions
other compounds also activate. For example, they may act, like
the proteases, along the chain of reactions involved in the feeding
reflex. Perhaps lactic acid, under their conditions affected some
step of the response. And there still remains the possibility that
Hydra has more than one receptor. All I can say is that in Hydra
littoralis all the factors that I mentioned in my talk influence the
response, and that there is no question that reduced glutathione is
a natural activator.

232 THE BIOLOGY OF HYDRA : 1961

BURNETT: We once found that dilute concentrations of bovine
testes hyaluronidase stimulated the feeding response. At that time
we assumed that our enzyme preparation was contaminated with
glutathione. A more recent preparation consisting of crystals quite
different from our original preparation was not effective.

LENHOFF: These are factors that you have to consider. First
you must dialyze to remove endogenous glutathione. For example
we found some of our enzyme preparations elicted a response before
dialysis but not afterwards.

KLINE: Some compounds may cause the mouth to open without
producing the true feeding reflex.

LENHOFF: Definitely. You can get mouth opening, but not the
true feeding reflex from many compounds, usually toxic ones. As
Dr. Loomis pointed out in his original paper, the best proof that a
compound can initiate the feeding reflex is to give the Hydra some
inert material impregnated with the compound you are testing. If
the Hydra ingests the inert material, then a true feeding reflex was
elicited by that compound.

The Nutrition of Hydra

David L. Claybrook

Dcpt. of Phijsiolo^ij 6- Fliarmacologij , Wayne State Universitij College of Medi-
cine, Detroit, Michigan.

The study of hydra nutrition is in its infancy. In fact, we are
not aware of any investigation of specific nutrient requirements
prior to our own. I suspect that the absence of such studies has been
due to the complexity of the prol^lem rather than to a lack of
appreciation for its importance. The laboratory culture of hydra
was more of an art than a science until Dr. Loomis' fundamental
research defining environmental conditions for optimal growth
( 12 ) . The numlier of environmental variables was then greatly
reduced to the point that the rate of growth coidd be directly con-
trolled by limiting the food supply.

The hydra's apparent refusal to ingest non-living food made it
essentially impossible to feed a formulated diet. When the gluta-
thione control of the feeding reaction was revealed (3), it offered
a means for feeding to the animals particulate preparations of the
experimenter's choice. With these possibilities in mind, we began
a study of hydra nutrition.

We undertook this investigation for two main reasons. First,
we wanted to know to what extent the hydra's requirements were
similar to and different from those of other animals. With the
exceptions of the protozoa and insects, very little work has been
devoted to the nutrition of invertebrates. Information on coelen-
terate nutrition would contribute significantly to our knowledge of
comparative biochemistry.

The second, and primary purpose of the project was to in-
crease the usefulness of the hydra as a biological material for the

iln part from a dissertation submitted to the Graduate School, The University of
Texas, in partial fulfillment of the requirements for the degree of Doctor of Phi-
losophy, August, 1960.

233

234 THE BIOLOGY OF HYDRA : 1961

chemical study of development and differentiation processes — a
field to be discussed by Dr. Eakin. Since the nutritional state of
an animal affects all of its physiological processes to some extent,
it is desirable to be able to control the nutritional state during the
study of other physiological phenomena. The development of chem-
ically defined nutrient preparations in which cultures of hydra or
hydra cells could be grown aseptically would give the investigator
complete biochemical control over the organisms.

Our ultimate goal was the propagation of hydra cells in a
chemically defined medium. With this sytem, we should be able
to determine the role of each tissue layer in processes such as
regeneration and sexual differentiation. However, we chose to begin
our experimentation with whole animals for two reasons: in general,
the requirements for cell propagation are much more critical than
those for growth of the intact organism. In the intact animal, speci-
fic trace nutrients may be supplied by specialized cells. There is also
a more rapid loss of essential nutrilites to the external solution
from the isolated cell. We hoped to discover approximate require-
ments before proceeding to precise studies at the cellular level.
Secondly, techniques for quantitative study had already been de-
veloped for whole hydra but not for dissociated cells. Thus the
nutritional value of an experimental diet could be determined by
its effect on an observable physiological process such as asexual
growth.

Our stock Hydra clone was obtained from a locally-isolated
strain of Hydra littoralis, and was grown according to the methods
of Ham and Eakin ( 1 ) , When fed daily with an excess of freshly-
hatched Artemia larvae, the Hydra grows at a maximum logarith-
mic rate. Presumably the animal receives an excess of all exogenous
requirements, and the limitation of growth rate is due to necessary
metabolic conversions within the cells. If the exogenous supply of
a growth factor is reduced below the maximum utilizable by the
animal, a reduction in the observed rate of growth should result.

In the search for a non-living diet, it was found that heat-
killed Artemia would support asexual growth of Hydra for at
least six months, but at a rate significantly below maximum. Al-
though the killed Artemia contained adequate amounts of reduced
glutathione to stimulate the feeding reaction, the solution had to be

DAVID L. CLAYBROOK 235

stirred gently to bring them into contact with the Hydras tentacles.
The effect of the period of heating on the subsequent growth
rate is depicted in Table 1. The reduction in the growth rate is
seen to be progressive with time of heating. This indicated to us
that some substance was being inactivated by the heat treatment
so that its availability to the Hydra became limiting to the growth
process.

TABLE 1

Relation of growth rate of Hydra littoralis. Ham strain, to period of

heating of Artemia (70°)

Growth Rate

1.9

3.3

3.5

4.5

5.2

U- =

hi 2
T

Heating time Doul:)hng time Growth Rate Constant

(min.) (days) (/c)**

.36
.21
.20
.15
.13

On the assumption that replacement of the growth-limiting fac-
tors to a nutritionally deficient diet would increase the rate of
]:)udding, we assayed numerous biochemical and biological sub-
stances for their capacities to stimulate budding when added as
supplements to a diet of heated shrimp. Artemia heated for 7
minutes at 70 were fed to Hydra cultures for at least a week
before the Hydra were used for bioassays. This period served to
deplete the animals of any reserve of the growth factor.

The heated Artemia diet was first supplemented with defined
and complex substances dissolved in the salt solution, bathing the
Hydra. Natural extracts, vitamins, amino acids, and other possible
growth factors, alone and in various combinations, were tested in
this system. No stimulatory effect on the growth rate was observed
in any expriment.

Since the lack of growth response to external supplements
could have been due to relative impermeability of the ectodermal
cells to dissolved materials, it was necessary to devise a technique
for introducing the test materials directly into the coelenteron

236

THE BIOLOGY OF HYDRA : 1961

where normal absorption could take place. A diagrammatic pic-
ture of the apparatus which was designed to inject a measured
volume into the individual organism is shown in Figure 1. The
apparatus features a micrometer-driven micro-liter syringe for de-
livering quantities in the micro-liter range.

^) ^ fe

to foot switch
for motor

to foot pedal
for release bar

Fig. 1. Micro-injector for feeding Hyc/ro.

In our standard injection test, adult Hydra without buds were
selected from the cultures maintained on heated Arteniia. The
animals were placed in 9-depression spot plates in large Petri
dishes, and each one was force-fed 0.2 [xl from a glass capillary
containing semi-solid agar in which the experimental diet was
dissolved or suspended. Twenty-four hours after injection, the ani-
mals were examined under a dissecting microscope, and the num-
ber of new buds in each dish of nine Hydra was recorded and
compared with that of the unfed control dish.

The relation of growth response to the quantity of material
injected is shown in Figure 2. The response was proportional to the

DAVID L. CLAYBROOK

237

"^^h

1.0
0.8
0.6

BUDDING RESPONSE TO INJECTED FRACTIONS

•

Woter- insoluble Fraction of Liver

Water-soluble, non- diolyzoble

Fraction of Liver

Water- insoluble Fraction

of Artemio ^

'^''^ ^

'^--'^ y

' 1

1 1 1 1 1 1 1 1 1 1 1

hydro

(Average of

9 Replicates) 0.4

0.2

I 2 4 6 8 10 20 30

^^/hydro
Fig. 2. Budding response to injected fractions.

logarithm of the dosage in some cases, while in the other experi-
ments log responses were not observed.

The relative potencies of some natural materials showing ac-
tivity in this system are listed in Table 2. The potencies on a dry

TABLE 2
Relative activities of natural supplements for promoting budding in Hydra

Potency

Material

(dry weight)

Bovine liver acetone powder"

Mouse liver

Mouse kidney

Mouse heart

Chick embryo extract

Escherichia coll

Dried yeast

Chlorella ellipsoiclea

Bovine liver extract,

non-dialyzable fraction

'Used as standard and assigned arbitrary value of 10.

238 THE BIOLOGY OF HYDRA : 1961

weight basis are expressed relative to an arbitrary standard, bovine
liver acetone powder. Activity was found in micro-organisms as
well as in crude mammalian tissues. Substances with no demon-
strable activity when fed internally included vitamins, amino acids,
protein digests, nucleic acids, carbohydrates, and microbiological
media.

An active soluble preparation, bovine liver extract, was sub-
jected to a number of physical and chemical tests in an effort to
characterize the active constituents. The results of such tests are
shown now in Table 3. The activity was found to be non-dialyzable,

TABLE 3
Potencies of modified non-dialyzable soluble extract

Fraction of

Treatment

Total Solids

Potency

Unmodified non-dialyzable extract

1.00

Ashing

0.02

Heating ( 2 hours, 70° )

Soluble fraction

0.20

Precipitate

0.80

Trypsin digestion

1.00

1-2

and was destroyed by ashing, characteristic of an organic macro-
molecule. Heating in solution precipitated but did not destroy the
active material. Incubation of the extract with trypsin or chymo-
trypsin resulted in the disappearance of nearly all biological ac-
tivity. The ultra-violet absorption spectrum of the soluble extract

TABLE 4
Potencies of ammonium sulfate fractions of non-dialyzable soluble extract

Ammonium

Sulfate*

Fraction of

Fraction

Total Solids

Potency

1.00

0-33%

0.28

33-66%

0.50

66-100%

0.13

Soluble at

100%

0.09

'Fraction precipitated between the indicated points of saturation.

DAVID L. CLAYBROOK

239

showed a peak absorption near 280 m/x, and the optical density
per milhgram of extract indicated a high percentage of protein.
All evidence, then, indicated that the active species were included
in the protein fraction.

Fractionation of the active extract with ammonium sulfate (Ta-
ble 4) revealed that all activity was salted out, but was distributed

Buds

0.6 -

0.4 -

0.2 -

0.0

Incubation Time in Hours at 37°C

Fig. 3. Effect of period of incubation with chymotrypsin on growth pro-
moting activity of non-dialyzable soluble extract.

among several fractions. While supporting the conclusion that the
active components were proteins, this data showed that the activity
was apparently common to several classes of protein.

The rate of inactivation of the extract by chymotrypsin is shown
in Figure 3. From this curve it would appear that intact protein
molecules, or relatively large fragments of them, are essential to
activitv in this extract.

240 THE BIOLOGY OF HYDRA : 1961

It is interesting to note that all purified proteins which have
been assayed were inactive in this system. These include casein,
bovine albumin, insulin, hemoglobin, and six bovine plasma frac-
tions. The wide distribution of activity in crude protein fractions,
contrasted with the absence of detectable activity in highly purified
proteins, suggests that the growth-stimulating factors could be small
molecules bound firmly to crude protein, but removable by repeated
purification. The evidence at hand has not enabled us to identify
the Hydra growth-promoting principle with any specific previously
recognized growth factors for other organisms.

While the micro-injection technique has been a very useful
method in the initial investigation of nutrition, it is still a tedious
procedure because of the individual attention required for each
Hydra. The mass culture of intact animals on a defined diet would
obviously require different methods. It appears from consideration
of other tissues cultured in vitro that the absolute biochemical re-
quirements can be determined only by study at the cellular level.
With the current progress toward maintaining coelenterate cells
in vitro, the time may be near when hydra cells may be used in
nutritional research.

I think the significance of our own experiments lies not in the
determination of specific nutrient requirements, but in the demon-
stration that Hydra can live and grow on a non-living diet, and
that nutrition of Hydra can be studied quantitatively by its effects
on a measurable physiological process — namely the asexual growth
process. Although we have only made a start toward understand-
ing the nutrition of Hydra, we are encouraged to believe that it
is a step toward developing the full potential of hydra as an experi-
mental system.

REFERENCES

1. Ham, R. G., and R. E. Eakin. 1958. Time sequence of certain physiological

events during regeneration in hydra. /. Exp. Zool. 139: 33-54.

2. LooMis, W. F. 1954. Environmental factors controlling growth in hydra. /. Exp.

Zool. 126: 223-234.

3. LooMis, W. F. 1955. Glutathione control of the specific feeding reactions of

hydra. Ann. N.Y. Acad. Sci. 62- 209-228.

DAVID L. CLAYBROOK 241

DISCUSSION

STREHLER: Do }'Ou need to include any particles along with
these soluble proteni fractions that were capable of supporting
growth?

CLAYRROOK: Well, our solutions were centrifuged for six hours
at 33,000 g, which means that any sur\iving particles must have
been rather small.

LENHOFF: I think what Dr. Strehler is getting at is that perhaps
the protein is being coagulated in the gut and is being engulfed as
particles. We have some evidence that H. littoralis gastrodermis
takes up mostly particles and leaves free amino acids behind in
the gut (Lenhoft, H. 1961. Exptl. Cell Research, 23: 335-353). Thus,
maybe the proteolytic enzymes destroy the growth-promoting prop-
erties of the heat-labile protein by reducing it to a non-particulate
solution of free amino acids that cannot be taken up by the gastro-
dermis.

CLAYRROOK: We don't know what happens after it gets inside
the gut.

GOREAU: What is Hydras digestive juice made out of?

CLAYRROOK: I have no information on this. Do others?

LENHOFF: We have fed about a million H. littoralis with shrimp,
until we knew, by other measurements, that the food was mostly
taken up by the gastroderm. Then we forced the Hydra to re-
gurgitate, took the extract, and precipitated it with 80% ammonium
sulfate. We found that there was proteolytic activity at pH 2.5 and 7.
These proteolytic enzymes probably aid in degrading the cells into
particles. But I doubt that the extracellular enzymes degrade the
particles all the way to free amino acids, because the particles,
when small enough, are rapidly phagocytized by the gastroderm.

GOREAU: The reason I ask is that Claybrook's very lovely
method allows one to withdraw things as well as introduce them,
and I was wondering if one could do microchemical analyses on
contents of the gut of the animal during various stages of digestion?

CLAYRROOK: I haven't tried this at all. I don't know.

242 THE BIOLOGY OF HYDRA : 1961

KLINE: When you maintain the Hydra on heat killed Artemia,
does the growth rate remain constant, even if reduced?

CLAYBROOK'. Fairly constant. It varies slightly with the various
lots of shrimp.

KLINE: Then you didn't totally destroy something that is needed.
Perhaps you reduced its concentration. How did you interpret the
results?

CLAYBROOK: The growth factor is not completely destroyed,
but becomes limiting to growth.

KLINE: In one experiment you had heat precipitated material
on which the Hydra were able to grow quite well.

CLAYBROOK: Right. This is the liver extract. We have not
fractionated shrimp because the relative supply of liver and shrimp
are not the same.

LENHOFF: Is it possible that the more you heat the shrimp,
the more the shrimp's cellular integiity is destroyed? And when
you put these damaged shrimp in water, essential factors leak out?
A few years ago Dr. Loomis and I were able to grow Hydra
on frozen shrimp, but had no success with boiled shrimp. We
thought then that boiling either destroyed a heat labile factor or
allowed essential heat-stable factors to leak out.

CLAYBROOK: It is possible, but in a few experiments we
found no activity in the supernatant that the shrimp were boiled
in. I wouldn't say this was conclusive.

LENHOFF: Was this supernatant solution either ninhydrin or
protein-positive?

CLAYBROOK: We didn't check at this stage but I'm sure that
there were ninhydrin-positive components there.

GOREAU: What I am speculating on now assumes a nervous
system! Living AHemia may be required because the struggle with
the prey could set up a reflex which causes hydra to secrete
enzymes or produce preabsorptive changes in the gastroderm,
which would allow digestion to proceed in a much more complete

DAVID L. CLAYBROOK 243

manner. The point is this. Perhaps the animal needs to struggle
with its prey? This is, of course, a complete speculation but we
may be dealing here with a phenomenon on the physiological
rather than biochemical level.

CLAYBROOK: I haven't tried any experiment which would
answer your question.

LENHOFF: Didn't Hijdra grow well on frozen shrimp?

CLAYBROOK: They grow at a reduced rate.

LENHOFF: At a very reduced rate?

CLAYBROOK: Not very reduced. But below that found with live
shrimp. The answer to this may also be leakage from the shrimp.

LENHOFF: But they do grow on frozen shrimp. I would think
that this would answer Dr. Goreau's speculation l^y showing that
the struggling of live prey is not required.

LOOMIS: It is interesting that apparently no carbohydrate is
necessary. In other words, pure protein is enough.

CLAYBROOK: Let's say carbohydrate is not limiting at this state.

LOOMIS: But you feed them solely on the 0-66% ammonium
sulfate fraction of liver protein?

CLAYBROOK: This alone will not support continued growth.
This is only a specific assay for the heat labile factor.

EAKIN: I think that some of you were not able to hear Dr.
Claybrook clearly when he described his method for demonstrating
the requirements of Hydra for the heat-labile factor. The Hydra
which he used as test organisms had been cultured at a sub-optimal
level of nutrition by feeding them on heated brine shrimp. The
response we studied was that of boosting them from this bare
maintenance level to that which we get when they are fed live
brine shrimp.

LOOMIS: It is a specific assay for the heat-labile factor?

EAKIN: That's right. Even the poorly growing controls are get-
ting a highly complicated diet in the heated Artemia.

244 THE BIOLOGY OF HYDRA : 1961

LOOMIS: I have often tried to get micro-pipettes into the mouths
of H. littoralis and out again without having them then regurgitate
what I put in their stomachs. Perhaps you open their mouths
with gkitathione?

CLAYBROOK: No, I force it open.

LOOMIS : Maybe that's the secret!

CLAYBROOK: Yes. Then I wait until he closes his mouth on
the pipette before injecting the material.

LOOMIS : And then it is water-tight as you pull out?

CLAYBROOK: The semi-solid consistency of the medium is es-
sential here. You can't use a liquid.

LOOMIS : How much agar do you use?

CLAYBROOK: I use 0A%, which is relatively thick. The Hydra
closes its mouth when the pipette is withdrawn and the viscous
solution remains in the gut.

LOOMIS : Will it flow down a microcapillary?

CLAYBROOK : Yes, if under pressure.

Isolation and Maintenance

in Tissue Culture of

Coelenterate Cell Lines

John H. Phillips

Department of Baeteriolofiij, University of California, Berkeley, California

The in vitro cultivation of coelenterate tissues has been reported
before (1, 2). However, attempts at the maintenance of such cul-
tures for prolonged periods of time and serial transfer of cultured
material have not apparently met with success. In addition, the evi-
dence in support of true multiplication of cells has not been entirely
convincing. The methods that will be discussed have led to the
establishment of cell cultures from the anemone Anfhopleura ele-
gantissima. These cultures have been transferred twenty to thirty
times and have been under in vitro cultivation for more than a
year. In addition, eight of the cell lines have been through one
single cell cloning. The resulting clones of eight to thirty-two cells
have given rise to cultures containing 10*^ to 10" cells.

First will be described the procedures which have been used
in isolation, cultivation and cloning of the cells, and this methodol-
ogy will be followed by a description of the cells and some of their
properties.

A somewhat more detailed account of methods will soon be
published (5). All glassware and rubber stoppers were cleaned
by autoclaving in 0.1% NaoCO... (4). Glassware was wrapped in
aluminum foil and sterilized by dry heat. Rubber stoppers were
autoclaved in large Petri dishes. All nutrient solutions were steri-
hzed by filtration, using Millipore filters. The nutrient medium
that has been found to be most useful consists of 0.7% Edamine\

1 Sheffield Chemical Company, Inc., Norwich, N. Y.

245

246 THE BIOLOGY OF HYDRA : 1961

an enzymatic digest of lactalbumin, in 90 /f artificial sea water (3)
containing 500 units of penicillin and 0.5 mg. streptomycin per ml.
Growth can be obtained over a range of Edamine concentrations
from 0.4 to 1.5% and artificial sea water concentrations of 40% to
100%. Yeasts and molds which are not inhibited by the antibiotic
mixture have at times presented difficulties. Mycostatin has been
used at a cencentration of 50 units/ml. to free cultures of these
contaminants. This antibiotic has not been included routinely be-
cause it appears to be somewhat toxic to the anemone cells.

The cell suspension used for the initial isolations is prepared
by mincing the animal in a beaker with a pair of scissors. In
some cases, lysozyme ( 1.5 mg./ml. of animal) was added to degrade
the mucus that is secreted. Approximately five volumes of arti-
ficial sea water is added per volume of minced tissue, and the
mixture is stirred briefly. It is allowed to stand in an ice bath for
approximately five minutes and filtered through two layers of
cheese cloth. The filtered suspension is freed of large tissue frag-
ments by centrifugation at 5 for 30 seconds at approximately 1000
g. The cell suspension containing very few tissue fragments is then
centrifuged as above for 10 minutes. The cells are resuspended in
sterile artificial sea water and the differential centrifugation is
repeated. The cells are finally washed three more times with
sterile artificial sea water. The last washing employs artificial sea
water to which has been added antibiotics at the above-mentioned
concentration. The cell suspension is diluted to a concentration
of close to 3 X 10' cells/ml. This corresponds to 0.100 O.D. at
660 m/x in the Beckman Spectrophotometer Model DU and is about
equal to 100 [ig. of cell protein/ml. or 5 X 10~^ ml. of packed
cells/ml. One-tenth ml. of such a suspension is used as the culture
inoculum. Figure 1 shows the appearance of such a suspension.
There is great heterogeneity of cell type, and the outstanding con-
taminant appears to be fragments of fibrous material from the
mesoglea. The two comet-shaped objects in the center of Figure 1
are this material. The cells show a size range from 8—2 /x.

The inoculum is placed in either a 60 mm Petri dish or into
a test tube of 15 X 130 mm. containing a piece of coverslip 10 X 20
mm. Five ml. of nutrient solution is added and mixed with the
inoculum. The test tubes are slanted to allow the cells to settle

JOHN H. PHILLIPS 247

<»

Fig. 1. Suspension of cells obtained from A. elegantlssima. Stained with
periodic acid Schiff's. Magnification 900x.

on the piece of coveislip. Either kind of preparation is incubated
at 15°. The cultures are examined microscopically at a magni-
fication of lOOx. Figure 2 shows well-developed clones growing on
the side of a tube culture. The piece of coverslip may be removed
from such cultures and used for more detailed examination. Grow-
ing cultures can be maintained in tubes for prolonged periods of
time, provided that fresh nutrient solution is added at weekly
intervals. A suspension of cells can be obtained for transfer to
new cultures by simply scraping some of the growth from the
glass surface with a sterile spatula or through the use of lysozyme
1.5 mg./ml. in 0.3 M ethylene diamine tetracetic acid adjusted to
pH 8.3 with NaOH. In either case, the final dispersal of the
clumps of cells requires agitation. Generally, the suspension is
drawn back and forth through a pipette. Cell suspensions may be
standardized as indicated above; however, complete dispersal is
generally not attained. The isolation of clones developing from

248

THE BIOLOGY OF HYDRA : 1961

.♦ .•>

Fig. 2. Clones of A. elegantissima growing on the side of a test tube.
Magnification 129x.

single cells is generally made difficult by the slight movement of
cells over the surface of the glass. Therefore, cloning procedure
of Puck (7) is generally used. Cells are mixed w^ith 10 ml. of
nutrient medium containing 0.2 /y agar, and the mixture is placed in
a Petri dish containing a layer of 1% agar in artificial sea water.
Developing clones are observed as clusters of cells that are generally
separated from one another by a distance equal to the cell diameter.
Development from a four through a thirty-two cell stage can be
observed. The generation time is somewhat in excess of twenty-
four hours. The clone can be removed with a capillary pipette
and transferred to fresh nutrient solution. Because of the dis-
tinctive appearance of a developing clone, there is no difficulty in
avoiding clumps of cells which were present in the inoculum.
The appearance of the cells growing in vitro shows certain

JOHN H. PHILLIPS 249

Fig. 3. Twelve-day-old clone of cells from A. elegantissima.
Stained with periodic acid Schiff's. Magnification 900x.

peculiarities, some of which it is hoped may be corrected through
the use of a better nutrient medium. Suspensions of single cells
obtained either directly from animals or from cultures do not
show reaggregation. On the contrary, a developing clone generally
shows outgrowth and separation of cells from the growing center.
The separated cells occasionally move a short distance before
becoming new centers of growth.

Figure 3 shows a twelve-day-old clone developing on a cover-
slip. The preparation was fixed in methanol and stained with
periodic acid Schiff's stain (5). The cells are filled with a granular
material that makes observation of the nucleus very difficult. These
granules, when observed in living cells by phase contrast micros-
copy appear as barred objects resembling mitochondria. Similar
intracellular structures can be observed in suspensions of cells
obtained directly from the animal, but such cells do not show as
high a concentration of these objects. When these cultured cells
are removed to artificial sea water containing ethylenediamine
tetraacetic acid (EDTA), they rapidly change their appearance to
that shown in Figure 4. The addition of sodium acetate to 0.1%
Edamine medium appears to produce a similar effect which is
under investigation at the time of this writing. Until the concentra-

250 THE BIOLOGY OF HYDRA : 1961

Fig. 4. Cultured cells washed with artificial sea water. Stained with
periodic acid Schiff's. Magnification 900x.

tion of these particles can be controlled, observation of mitosis
in developing clones is impossible.

The cells, particularly those toward the center of the clone
in Figure 3, are surrounded by a red staining, carbohydrate-con-
taining material which apparently acts as an intercellular cement.
It can be weakened by both lysozyme and EDTA, but these agents
even in combination do not result in complete separation of the
cells. Since lysozyme functions as a i8(l-^4) N-acetyl hexosamini-
dase (8), the presence of this carbohydrate derivative in the
material appears likely. The material is not susceptible to the
chitinase of Helix pomatia, hyaluronidase, nor trypsin. The action
of EDTA suggests either the presence of bridges formed by diva-
lent ions or possibly the activation of the lysozyme-like enzyme
that has been detected in the secretions of these animals (6).
Pollak's trichrome stain (9) has also been used in studies of this
material. It is again stained red. This staining reaction is given
by elastic fibers. Mucus assumes a green coloration by this stain-
ing procedure. It appears likely that the material in question is
other than mucus. Until the nature of this material is better under-
stood, the methods for its degradation are available, quantitative

JOHN H. PHILLIPS 251

work— for example, the accurate determination of generation
time and cloning efficiency— is made difficult.

The ease with which cell lines from this anemone can be
established and maintained in the laboratory is encouraging. It
will be of interest to determine if the cells of other coelenterates
behave in a similar manner.

These studies were supported l:)y grants from the National Science Foundation
and the United States PubHc Health Service.

REFERENCES

1. Gary, L. R. 1931. Report on invertebrate tissue culture. Carnegie Inst. Wash.

Yr. Bk. 30: 379-381.

2. Lewis, M. R. 1915-1916. Sea water as a medium for tissue cultures. Anat. Rec.

10: 287-299.

3. MacLeod, R. A., E. Onofrey and M. E. Norris. 1954. Nutrition and metabo-

lism of marine bacteria. 1. Survey of nutritional requirements. /. Bad. 68:
680-686.

4. Madin, S. H., p. C. Andriese and N. B. Darby. 1957. The in vitro cultivation

of tissues of domestic and laboratory animals. Amer. J. of Vet. Res. 69:
932-941.

5. Phillips, J. H. In vitro maintenance and cultivation of cells from marine

invertebrates. Methods in Medical Research (in press).

6. Phillips, J. H. Immune mechanisms in the Phylum Coelenterata, Second Annual

Symposium on Comparative Biology. The Lower Metazoa: Comparative
Biology and Phylogenij. To be published by Academic Press, N. Y.

7. Puck, T. T., P. I. Marcus and S. J. Cieciura. 1956. Clonal growth of mam-

malian cells in vitro. ]. Exp. Med. 103: 273-284.

8. Salton, M. J. R. and J. M. Ghuysen. 1959. The structure of di and tetra sac-

charides released from cell walls by lysozyme and streptomyces Fi enzyme
and the y8(1^4) N-acetyl hexosaminidase activity of these enzymes.
Biochim. et Biophys. Acta 36: 552-554.

9. Sano, M. E. 1949. Trichrome stain for tissue section, culture, or smear. Amer.

J. Clin. Path. 19: 898.

DISCUSSION

MUSCATINE: Was the animal kept in artificial sea water?

PHILLIPS: Our artificial sea water preparation is capable of
maintaining the intact animal for a long period of time, but they
are normally kept in real sea water.

MUSCATINE: Is there any particular criterion that you use for
the well-being of the animal?

252 THE BIOLOGY OF HYDRA : 1961

PHILLIPS: No. It just continues to look healthy.

GOREAU: That is a beautiful piece of work. Do you know
what cell types your cultures actually come from? Have you tried
adding zooxanthellae?

PHILLIPS: I haven't tried adding zooxanthellae. Some people
at Stanford are interested in this problem. I am planning to give
them my cultures to do this. With respect to the cell that I have
growing in culture, this becomes an extremely difficult question to
answer. For one thing, the appearance of the cells growing in
culture may be markedly different from the cells that one sees in
the intact animal as all the cells tend to round up on being freed
from the tissue mass. This makes it impossible, on the basis of
cell shape, to decide whether it is endoderm, mesoglea, or ectoderm.

GOREAU: Perhaps you could start your cultures with scrapings
from specific areas rather than the whole animal.

PHILLIPS: This is something we want to try. I have not devoted
a great deal of work to these culture lines although I've had them
in the laboratory for sometime.

GOREAU: A very important matter to anyone who has ever
tried to dissect living coelenterates is the horrible problem of
being flooded with mucus. Are you actually cutting this down
with lysozyme?

PHILLIPS: Definitely. There is one trick to that. The lysozyme
should not be added to sea water. High electrolyte concentration
is quite inhibitory to the action of lysozyme. It decreases its activity
by almost 50%. That's the reason I add it directly to the animal
before mincing the tissue.

There is another thing I should mention, namely, the use of
fluorescent antibody techniques for identification of materials with-
in tissue. I have carried out work of this sort with these cells using
rabbit anti-anemone serum and fluorescently labeled dog anti-
rabbit globulin serum. This leads to a nice fluorescent uptake by
the cells growing in culture, and it also results in an uptake of
fluorescence by whole cell suspensions. But, I would not care to
put this forth as anything but supporting evidence for these cells

JOHN H. PHILLIPS 253

being from the anemone. I think this proof must come from repeated
isolations, such as we carried out, and from a comparative study
of the morphology of the cells. Also a consideration of the cloning
efficiency assists in discarding the possibility that the cultured cell
is some parasite present in small numbers within the animal.

WAINWRIGHT: Have you tried collagenase on the intercellular
material?

PHILLIPS: No. Those are the only enzymes I have tried so far.
It is resistant to trypsin and hyaluronidase but degraded by lyso-
zyme.

WOOD: I was not quite clear about your statements concerning
the mitochondria. Have you tried a specific mitochondrial staining
technique or do you have other criteria for identification?

PHILLIPS: No. I simply said that they resembled mitochondria
in that they were markedly bar shaped. That's all.

STREHLER: Is there only one morphological type of cell?

PHILLIPS: One sees a variety of cell types in developing cul-
tures. For example, the ratio between nuclear and cytoplasmic size
varies as well as the distribution of the granules within the cells. At
the same time clone cultures derived from a single cell also shows
this variation.

SLAUTTERBACK: If the anemone is anything like hydra, you
can determine whether or not they are gastroderm cells by expos-
ing the animal to a thorotrast solution for a short time. Thorium
dioxide serves as an excellent tag because only gastroderm cells
pinocytize it.

WOOD: Could you be certain that free cells derived from ecto-
derm would not pinocytize or phagocytize some thorotrast?

PHILLIPS: These cells do show a rapid uptake of such ma-
terials as bovine and human serum albumin. If one labels such
proteins with azo dyes within 15 minutes you get cells with
brightly stained inclusions and the cells remain colored for long
periods of time. In fact, it was in connection with immunological
studies that I first became interested in cultivating these cells.

254 THE BIOLOGY OF HYDRA : 1961

SLAUTTERBACK: In response to Dr. Wood's comment, my
suggestion was that the animal be exposed to the colloidal thorium
dioxide before it was cut up. In that case there would be no
thorium in the ectoderm cells.

PHILLIPS: True, if the label remains.

PASSANO: What is the chromosomal integrity in your clones
over a period of time?

PHILLIPS: I don't know. Until I can get rid of these granules
and control their formation I do not want to even attempt to ob-
serve mitotic figures.

STREHLER: Does colchicine block their mitosis?

PHILLIPS: I have not tried it yet.

Symbiosis in Marine and
Fresh Water Coelenterates

Leonard Muscatine

Laboratories of Biochcmistrij, Howard Hughes Medical Instittttc, Miami, Florida

In studying the significance of symbiotic algae for the nutrition
and growth of their invertebrate hosts, we have been guided by two
objectives : a ) to estabhsh the existence of a nutritional relationship
between algae and host, and b) to characterize the chemical basis
of this relationship.

Direct evidence for the contribution of carbon compounds from
symbiotic algae to the tissues of the host has been demonstrated
in a sea anemone ( 9 ) , a coral ( 3 ) , and in green hydra ( 5 ) .

In this paper, we demonstrate a direct relationship between algal
symbionts and changes in mass or growth of a marine and a fresh-
water coelenterate. Our data show that retarded weight loss, en-
hanced growth, and prolonged survival of the animals studied could
be attributed to the presence of symbiotic algae.

STUDIES ON SEA ANEMONES' -

Experiments demonstrating retardation of weight loss were con-
ducted on Anthopleura elegantissima (Brandt, 1835), an intertidal
anemone which contains zooxanthellae within its gastrodermal cells
(Fig. 1). Specimens without algae, found beneath fish canneries

^Part of a tliesis submitted in partial fulfillment of the requirements for the degree
of doctor of Philosophy, Department of Zoology, University of California, Berkeley.

-This investigation was supported by a fellowship (EF-9653) from the National Insti-
tutes of Allergy and Infectious Diseases, Public Health Service.

255

256

THE BIOLOGY OF HYDRA : 1961

^^'

-1^'-

Fig. 1. Electron micrograph of a transverse section through a musculo-
epitheiial cell of an anemone showing the intracellular location of an algal
cell, a) animal cell, b) algal cell, c) chromatophore. d) pyrenoid. (Prepared
with the assistance of Miss Jane Westfall)

at Pacific Grove, California, served as controls and are referred to
as albinos.

In order to evaluate quantitatively the effect of the algae on
the nutrition of the host, we measured changes in weight of normal
and albino anemones starved in light and darkness for 11 weeks.
Reduced weight, i.e., weight under water, was used to measure
weight changes. This method eliminates error from surplus fluid
and, in contrast to dry weight, allows repeated measurements on
living individuals.^

^There were no major changes in the specific gravity of the animals themselves
during the course of the experiment, showing that all of the observed changes were
true weight changes.

LEONARD MUSCATINE

257

Fig. 2. Arrangement of apparatus for rapid measurement of the reduced
weight of a sea anemone in sea water of known temperature and density.
The animal is suspended by a thin constantan wire hooked into its actino-
phorynx.

Two groups of five normal anemones were placed into aerated
containers of twice-filtered sea water at 14.0^ ± 1.5^. One group
was continually illuminated by 200 ft. c. of fluorescent illumination
( Champion— Warm White) while the other was kept continually
in darkness. Both groups were allowed to starve.^ The reduced
weight of each individual was measured (Fig. 2) at intervals of
four days or more and the sea water in all containers was renewed
weekly. Individuals in darkness were weighed in dim light. As
additional controls, two groups of five albino anemones were treated
in a manner identical to the normal svmbiotized anemones. Details

^Fed anemones were unsatisfactory experimental animals. Erratic behavior ( e.g. pre-
mature egestion, failure to feed) interfered with attempts to control feeding.

5.58 THE BIOLOGY OF HYDRA : 1961

NORMAL,

LIGHT

•

NORMAL,

DARK

K. 0

•

ALBINO,

LIGHT

ALBINO,

DARK

^ 5

•
•

-J

•

1 10

—

•
•

n
*

5 15

•

▲

•

▲

;:£ 20

—

▲ A

•

▲

•

5 25

•
•

•

•
•

•

Uj 30

▲

•

Ci:

1 A 1

1 1

0 1 23 456789 10 II 12

TIME IN WEEKS

Fig. 3. Chonge in reduced weight of normal and albino anemones starved
in light and darkness. The ordinate is the percent change from initial weight
and denotes weight loss.

of other methods in these experiments are given elsewhere (8).
Reduced weight changes of symbiotized and albino anemones
starved in light and darkness are depicted in Figure 3, and expressed
as percent change from initial weight vs. time in weeks. The results
show that durmg starvation all anemones lost weight at a near con-
stant rate, and that symbiotized anemones lost weight at about half
the rate of albinos. The possiliility tliat light could have directly

LEONARD MUSCATINE 259

affected weight loss was tentatively ruled out since albinos in light
and darkness lost weight at the same rate. We therefore conclude
that the lower rate of weight loss by symbiotized anemones is re-
lated to the presence of algae.

These observations, along with evidence from tracer studies (9),
suggest that during starvation carbon contributed by the algae,
together with host excretory nitrogen, is used for the synthesis of
organic compounds necessary for maintenance of weight. This view
emphasizes the possible secondary role of the algae in reclaiming
waste nitrogen.

STUDIES ON HYDRA

With the introduction of techniques for the mass culture of
Hydra (7) an opportunity presented itself for quantitative studies
on plant-animal symbiosis in the laboratory. Chlorohydm viridis-
sima/' a hydra containing zoochlorellae within its gastrodermal cells
( 14 ) , may now be grown under controlled environmental conditions
and in a fluid of known ionic constitution. Growth may be measured
in terms of protein or logarithmic increase in number of hydranths
(6). In addition, problems encountered with sea anemones, such
as removal of algae and erratic feeding behavior, are easily
resolved.

C. viridissima was routinely grown in our laboratory in the fol-
lowing culture medium ("M" solution): 10 "^M Tris (hydroxy)
methylaminomethane buffer, pH 7.6, lO'-^M CaCL, 10 'M
NaHCOs, 10-W KCl, and IQ-^M MgCL, in de-ionized water.

Algae-free C. viridissima were obtained by growing green indi-
viduals in M solution plus 0.5% glycerin (v/v) for 7-10 days,
following the original technique of Whitney (13). These albinos
then grew normally in M solution and did not regain an algal flora.

Growth studies. All growth experiments were conducted at
21°-23° using the method of Loomis (7). Ten hydranths (five uni-
form hydra, see ref. 4) from mass cultures were put in 30 ml.
of M solution in shallow Petri dishes placed four inches from a
single 40-watt fluorescent light ( Sylvania-Cool White). Daily, the

^Tentative identification.

260

THE BIOLOGY OF HYDRA : 1961

FED DAILY

GREEN

ALBINO

TIME IN DAYS

Fig. 4. Semi-log plot of growth rates of duplicate cultures of green and
albino C. yiridissima fed daily in the light.

number of hydranths was counted and then each hydranth was fed
on a dense suspension of Artemia nauphi. One hour after feeding
and again, six hours later, the culture medium was renewed. This
routine was followed for 5-7 days.

Figure 4 shows that green and albino C. viridissima, when fed
daily, have nearly identical logarithmic growth rates. These results
imply that the algae do not contribute anything to the host that
cannot be acquired from an exogenous food supply. Under optimal
conditions, nutritional benefit would not be expected to manifest
itself in terms of growth of the host because the maximum growth
rate (kmax), a property intrinsic to the species, cannot be exceeded,
regardless of the magnitude of the algal contribution. Therefore, we
conducted growth experiments in which the amount of food was
limited, reasoning that this would then allow benefit from the algae
to express itself. Figure 5 demonstrates that, when fed every second

LEONARD MUSCATINE

261

FED EVERY SECOND DAY

GREEN

t ALBINO

TIME IN DAYS

Fig. 5. Same as Figure 4 but fed every second day. Arrow denotes time of
feeding.

day, green liydra deviated only slightly from normal logarithmic
growth. But under the same conditions, albino hydra showed not
only a more pronounced deviation, but also required more time to
regain normal growth after feeding was resumed. More striking dif-
ferences appeared when these two groups were fed every third
day. Figure 6 illustrates the sharp decline in rate of budding by
both groups during the interval without food. But after feeding,
green hydra immediately resumed a normal maximum growth rate.
In contrast, growth of albinos lagged and did not return to normal.
The effect of complete elimination of food is shown in Figure 7.
Ten hydranths of each kind were removed from mass cultures and
starved in the light in 30 ml. of M solution in Petri dishes (4"
diam.). The culture medium was renewed once daily. Under these
conditions, green hydra continued to produce buds for 7-9 days and

262

THE BIOLOGY OF HYDRA : 1961

^ 80

FED EVERY THIRD DAY

GREEN ^

ALBINO t

J I \ \ \ L

TIME IN DAYS
Fig. 6. Same as Figure 5 but fed every third day.

survived an additional 7-10 days until gradual diminution in size
resulted in death. In contrast, albino C. viridissima, under these
conditions, stopped budding after 1-3 days; within the next six days,
all had disintegrated. These results show that the algae are essential
for prolonged survival under starvation conditions.

Early disintegration and death of albinos was unusual since a
characteristic of most species of hydra, including green C. viridis-
sima, is to gradually "waste away" when starved ( 1 ) . One explana-
tion of this death gives us a clue to a possible nutritional role of the
algae. Dixon (2) has stated that tissue death results from inability
to synthesize coenzymes. By removing algae from C. viridissima we
may have removed a source of coenzymes, or coenzyme precursors,
normally available from algae during starvation or from food during
normal feeding conditions. This idea fits well with results of limited
food experiments, where green hydra show maximum growth imme-

LEOXARD MUSCATINE

263

co20

^ 15
o 10

5 -

STARVED

•

—

•
o

• •

o o

1 1

TIME IN DAYS

Fig. 7. Bud production and survival of green (closed circles) and albino
(open circles) C. virid'issima starved in light. Each point represents the mean
number of hydranths in duplicate culture vessels.

diately after feeding (Figs. 5, 6) suggesting that they are primed
with cofactors necessar\- for the effieient con\'ersion of crustacean
protein into coelenterate protein. In contrast, albinos showed a lag
after feeding. This lag may represent the time during which a
cof actor from food is mobilized.

These data take on special interest when compared to the
results of field studies on the nutrition of corals. Corals contain-
ing zooxanthellae grow optimally in spite of a low exogenous food
supply (10, 11, 12). Our results with C. viridissima suggest that

264 THE BIOLOGY OF HYDRA : 1961

symbiotic algae can account for this by promoting efficient utiliza-
tion of available food.

ACKNOWLEDGEMENTS

It is a pleasure to acknowledge the counsel of Drs. C. Hand and
R. I. Smith at the University of California, Berkeley. Studies on
hydra were initiated under the guidance of Dr. H. M. Lenhoff, to
whom I am indebted for help in all phases of this investigation.

REFERENCES

1. Brien, p. 1961. The fresh-water hydra. Amcr. Sci. 48: 461-475.

2. Dixon, M. 1941. Multi-enzyme systems. Cambridge, 100 pp.

3. GoREAU, T. F. and N. I. Goreau. 1960. Distribution of labeled carbon in rccf-

building corals with and without zooxanthellae. Science 131: 668-669.

4. Lenhoff, H. M. and J. Bovaird. 1961. A quantitative chemical approach to

problems of nematocyst distribution and replacement in Hydra. Detyelop.
Biol. 3: 227-240.

5. Lenhoff, H. M. and K. F. Zimmerman. 1959. Biochemical studies of symbiosis

in Chlorohydra viridissima. Anat. Rec. 134: 559.

6. LooMis, W. F. 1954. Environmental factors controlling growth in hydra. /. Exp.

Zool. 126: 223-234.

7. LooMis, W. F. and H. M. Lenhoff. 1956. Growth and sexual differentiation of

hydra in mass culture. /. Exp. Zool. 132: 555-574.

8. Muscatine, L. 1961. Some aspects of the relationship between a sea anemone

and its symbiotic algae. Ph.D. Thesis, University of California, Berkeley,
100 pp.

9. Muscatine, L. and C. Hand. 1958. Direct evidence for transfer of materials

from symbiotic algae to the tissues of a coelcnterate. Proc. Nat. Acad. Sci.
44: 1259-1263.

10. Odum, H. T. and E. P. Odum. 1955. Trophic structure and productivity of a

windward coral reef community on Eniwetok atoll. Ecol. Monogr. 25:
291-320.

11. Sargent, M. and T. S. Austin. 1949. Organic productivity of an atoll. Traits.

Amer. Geophys. Union 30: 245-249.

12. Sargent, M. and T. S. Austin. 1954. Biologic economy of coral reefs. U. S.

Geol. Survey Prof. Pap. 260E, pp. 299-300.

13. Whitney, D. D. 1907. Artificial removal of the green bodies of Hydra viridis.

Biol. Bull. 13: 291-299.

14. Wood, R. L. 1959. Intercellular attachment in the epithelium of Hydra as

revealed by electron microscopy. /. Biophysic. Biochem. Cytol. 6: 343-352.
See also p. 55, this volume.

LEONARD MUSCATINE 265

DISCUSSION

WAINWRIGHT: Is the number of hydranths proportional to the
total amount of protein?

MUSCATINE: Yes, the relationship is linear up to about 75 hy-
dranths.

WAINWRIGHT: Is this so in the starved experiment?

MUSCATINE: Preliminary experiments show that after 5 days of
starvation albinos consist of less protein per hydranth than the
greens.

EAKIN: Are you carrying along the colorless algae as a parasite
in your albino organism?

MUSCATINE: Microscopic examination after treatment with gly-
cerin indicates that algae are no longer present. We use only those
albinos which do not regain an algal flora when placed back in the
glycerin-free culture solution.

EAKIN: I will be reporting on an organism we developed by cul-
turing Chlorohijcha in the dark, one which we call "brown ChJoro-
hydra," and which undoubtedly corresponds to your "albino." We
have not been able to detect the presence of any colorless algae in
them.

BURNETT: One of my students, Peter Wernik, finds that albinos
take in more glycogen and protein reserve droplets than do green
hydra.

MUSCATINE: Do you feel that the green hydra use food more
efficiently than the albinos?

BURNETT: I don't know. Possibly the greens aren't requiring as
much; a hydra always takes in just about what he needs. Did I
understand you to say that your animals budded during 8 days of
starvation?

MUSCATINE: Yes, budding by green hydra persists for a week.
They double their number in this time.

GOREAU: What is the ratio of plant to animal biomass in Chloro-
hijdra?

266 THE BIOLOCY OF HYDRA : 1961

MUSCATINE: I have no information on the algae in Chlorohydra
yet. But I have good data for Anthoplcura clcgantissima where one
can determine the biomass of the alga flora by using quantative
pigment techniques.

GOREAU: You mean chlorophyll?

MUSCATINE: Yes, and the various carotenoids. Using the
method of Richards with Thompson, and using cell counts and dry
weight data from pure suspensions of zooxanthellae, we find that
in Antlwpk'ura, the ratio of animal to algae on a dry weight basis
is about 332 to 1.

GOREAU: Such data is very important in relation to turnover
studies.

CLAYBROOK: How critical is the magnesium requirement for
Chlorohydra? Is this essential or does it merely increase the growth
rate?

MUSCATINE: The maximum doubling time of green or albino
hydra is about 1.2 days. Without magnesium it is only 1.9 to 2.8
days.

LENHOFF: I think it's important to add that they reqidrc mag-
nesium in order to grow. When we first received our Chlorohydra,
we could not grow them on any of our other culture solutions. The
last cation that we tried was magnesium. Then they doubled nearly
every day.

CLAYBROOK: In our experiments with Chlorohydra, we don't
add any magnesium to the solution.

MUSCATINE: Well, there is a possibility that they get enough
in their food, or perhaps you have a different strain of animals?

CLAYBROOK: It could be.

EAKIN: Although we have maintained our Chlorohydra in syn-
thetic solutions to which we have added no magnesium (solutions
which give optimal maintenance conditions for Hydra littoralis), we
find that the addition of Mg+ + decreases the doubling time and on
occasions has caused clones showing signs of depression to return
to normal.

LEONARD MUSCATINE 267

LOOMIS: We grow them happily in 5% artificial seawater. We
make up an MBL artificial seawater with deionized water, not dis-
tilled. That's the main thing, no copper. In fact, we have nearly a
dozen hydroids growing in artificial seawater. Cordylophora grows
nicely in 10% MBL water while Chlorohydra grows in 55^ MBL. Of
course, that has magnesium in it.

I would like to make another point. Some day, somebody ought
to study how glycerine makes the endothelial cells spit out their
contained Chlorella. It would he interesting to study this incredible
reaction, as well as to try and reinfect albino green hydra with free
Chlorella. I don't think this has ever been done with green hydra,
or lichens either. In other words, you can separate; but no one has
yet recombined the two symbiotic forms that I know of.

SLAUTTERBACK: Regarding reinfection, I have taken eggs,
which as you know are white, from Chlorohydra and hatched them
separately. If the resulting albinos are returned to a culture of green
hydra, they remain white for about 2 weeks.

FULTON: I think some German workers have succeeded in re-
infecting white Chlorohydra, but not other species of hydra. Inci-
dentally, we found that the antibiotic chloramphenical cures green
hydra of the algae in a couple of days, much faster than glycerine.

MUSCATINE: What concentration was used?

FULTON: I'm not certain, but I think it was 200 /.ig. per ml.

AlUSCATINE: I tried various algicides with the anemones, and
neither the commercial product Algaedyne, which is a colloidal sil-
ver solution, nor a high concentration of Streptomycin, nor starva-
tion in darkness succeeded in totally ridding the animal of its algae.
When starved in darkness the animal becomes very small but still
retains algal cells which can be shown to increase in pigment con-
tent. This might be regarded as evidence for heterotrophic activity
in these zooxanthellae.

FULTON: Did you try chloroamphenicol?

MUSCATINE: No.

BURNETT: Have you e\'er noticed differences in the distribution
of the algae along the column?

26S THE BIOLOGY OF HYDRA : 1961

MUSCATINE: Yes, very often one sees regional differences. How-
ever, I am not sure of the significance of this.

BURNETT: I mention this because I had a chance to observe a
very interesting green hydra in Brien's laboratory. This animal ( H.
viridis) underwent what seems to be a somatic mutation. The
peduncle on this form resembles a stolon and is several times larger
than the gastric region. The whole animal may be one and a half
inches long, surprising dimensions for a green hydra. The peduncle,
unlike that of normal H. viridis, contains more algal bodies per cell
than the gastric region. Also food materials pass into this region in
greater amounts than into a normal peduncle. What is most inter-
esting is that this mutant form not only reproduces asexually by
budding but also by pinching off the distal portion of its peduncle.
This detached portion then regenerates into a complete organism.
It is marvelous!

EAKIN: Did you try increasing the oxygen tension while growing
the albino hydras?

MUSCATINE: Well, we're just getting into gas analysis. We have
conducted preliminary experiments growing green and albino hydra
in air plus 0.4% COo, but the results were not definitive. Eventually
we will control pCOo and pOo.

EAKIN: It will be interesting to see if the high oxygen tension
can reverse some of the effects observed in the absence of the algal
chlorophyll.

MUSCATINE: Yes, that's a good way to attack this, going through
the algae. I would also like to see if green hydra show an action
spectrum for growth rates which can be related to the absorption
spectrum of chlorophyll.

On the Relation of Calcification
to Primary Productivity in
Reef Building Organisms

T. F. GOREAU

Physiology Department, University College of the West Indies, Mona, Jamaieu,
W. I., and Department of Marine Biochemistry and Ecology, New York Zoological
Society.

Coral reefs are tropical shallow water communities built up by
calcareous organisms attached to the sea bottom. Such ecosystems
may be regarded as biochemical factories which catalyse a large
scale transfer of dissolved calcium and carbonate ions from sea
water into the sediments as insoluble calcium carbonate. The result-
ing reef limestones are deposited in typical formations which may
in time become several thousand feet thick, as for example in some
of the Pacific atolls ( 9 ) .

A unique characteristic of coral reefs, found in no other deposi-
tional system in the biosphere, is that maximum biological accre-
tion of calcareous matter takes place only in the turbulent surface
waters where the forces of mechanical and chemical erosion are
also at a maximum. Corals and algae which build reefs do so by
secreting hard calcareous masses that become aggregated into an
organised coherent structure adapted for maximum attenuation of
mechanical stresses set up by the constant battering of the seas,
yet so shaped as to expose a maximum surface area for efficient
matter-energy exchange with the environment. The papers of
Tracey ef al. (16) and Emery ct al. (1) should be consulted for
further aspects of this problem.

In the West Indies, the interlocking reef framework is built up
by the larger Scleractinia and Milleporidae, their separate colon-

269

270 THE BIOLOGY OF HYDRA : 1961

ies becoming cemented into a single nnit by lithothamnioid algae.
The finer, more \'oIuminous, lagoon and forward slope sediments
are produced chiefly by calcareous green algae, with Scleractinia,
Gorgonia, Foraminifera, sponges, mollusks, arthropods and ech-
inoderms contributing in xarious proportions depending on local
factors. Owing to its stability and exposure to the seas, the frame-
work is probably the site where most of the calcium carbonate pro-
duction of the reef occurs. Only a fraction of this is ultimately de-
posited in situ since the greater part of the calcareous material pro-
duced here is washed out by waves and redeposited in the calmer
water of the lagoon or the seaward slope.

A large proportion of the total biomass of coral reefs is due to
algae which grow in great abundance in all zones, ranging from the
shallowest parts of the rampart to depths exceeding two hundred
feet on the forward slope. The algal population of reefs can be divid-
ed into two categories: the free-li\'ing fleshy, filamentous, and cal-
careous algae; and the symbiotic unicellular zooxanthellae living
in coelenterates.

All reef-building Scleractinian corals without exception contain
zooxanthellae. So do most Hydrocorals, Actinaria, Zoanthidea,
Alcyonaria and Gorgonia living in reefs. According to the existing
nomenclature, those calcareous coelenterates which have zooxan-
thellae are said to be hermatypic, or reef-building; whereas those
species lacking zooxanthellae are said to be ahermatypic or non-
reef building. The former are limited in their vertical distribution
to the upper parts of the euphotic zone and never grow in
dark places. The ahermatypes are usually found in deep water be-
low the euphotic zone although some species occur in shallow wa-
ter where they tend to favour dark crevices. The basic difference be-
tween hermatypic and ahermatypic coelenterates is that the
former grow much faster to much larger sizes than the latter. Never-
theless, some ahermatypic corals are known under certain condi-
tions to form deep-sea banks which bear a superficial resemblance
to shallow^ water reefs ( 14) .

Although there is an absolute correlation between the pres-
ence of zooxanthellae in calcareous coelenterates and their ability
to build reefs, the relationship of the algae to their hosts and to
the bio-economy of the reef as a whole is not yet clearly under-

T. F. GOREAU 271

stood. The so-called "zooxanthella problem has been the sub-
ject of much controversy because some investigators ha\e
failed to recognise the multiplicity of host-symbiont relationships
in the different groups of coelenterates: ranging from total nutri-
tional dependence on zooxanthellae in some xeniid Alcyonacea
(2) to nutritional independence in the Scleractinia which are spe-
cialised carni^'ores (21). There can be little doubt that zooxanthella-
coelenterate symbioses have exolved independenth' in many unre-
lated groups at different times, thus accounting for the haphazard
variety of the association in the \ arious classes and orders of the
phylum. For further details and references regarding the zooxan-
thella problem, the papers of Yonge (19, 20), Vaughan and Wells
(17), Odum and Odum (10), and Goreau (4) should he consulted.

CALCIUM DEPOSITION AND PHOTOSYNTHESIS
IN REEF CORALS

Growth in corals is achie\ ed b\' an increase in mass of the cal-
careous skeleton and a concommittant proliferation of the overly-
ing tissues. Our recent underwater studies on reef corals suggest
that e\'en within any given species there may be no constant re-
lationship between these two kinds of growth and that colony shape
is to a certain extent controlled by ^'ariations in the ratio of new
skeleton to new tissue. To study the factors which regulate calci-
fication in corals and other calcareous organisms, we ha\e dexeloped
new methods for the fast quantitative assay of growth by the use of
radioactive tracers. Calcification is determined from the rate with
which Ca^"* ions added to the sea water medium is deposited into
the skeleton as Ca^' CO.,. under various conditions, e.g. light and
dark. The procedure, which has been described elsewhere (3, 6),
requires only a few hours; the experimental runs can be carried out
in the field, and growth gradients are determined by sampling
different parts of experimental colonies.

Our observations demonstrate that calcification in reef-build-
ing corals is dependent on the ambient light intensity to the ex-
tent that growth in foiuteen species tested is on the a\'erage ten
times faster in sunlight than in darkness (6). Calcification is re-

272 THE BIOLOGY OF HYDRA : 1961

duced by approximately fifty per cent on a cloudy day under other-
wise similar conditions. By contrast, the calcification rates of some
shallow water ahermatypic corals lacking zooxanthellae do not re-
spond significantly to changes in light intensity. The stimulant
effect of light on reef coral calcification disappears when the zoo-
xanthellae are removed by culturing corals in darkness for about
three months.

Inherent species specific factors, independent of the zooxanthel-
lae, also exert an important influence on calcium deposition.
One example of this is the growth gradient of ramose corals such as
Acropora cerviconiis where the large pale apical polyps that con-
tain relatively few zooxanthellae calcify several times faster than
the much smaller adjacent lateral corallities the tissues of which
are packed with large masses of zooxanthellae. The enzyme car-
bonic anhydrase also appears to play an important role in coral
calcification. We have found carbonic anhydrase activity in repre-
sentative species of all major groups of Coelenterata. The occur-
rence of the enzyme has no relationship to the calcareous habit,
or to the presence of zooxanthellae, which themselves do not
contain significant amounts of carbonic anhydrase. The treatment
of reef corals with a specific carbonic anhydrase inhibitor ( Diamox,
Lederle) results in an average fifty per cent reduction of the calci-
fication rate in the light, and a seventy five per cent reduction in
darkness. The effect of carbonic anhydrase inhibition on the calci-
fication rate is partially reversed in the light when the zooxanthel-
lae are photosynthesizing. It therefore appears that carbonic anhy-
drase and the zooxanthellae act in synergy to potentiate calcium
deposition in corals ( 3 ) .

The mechanisms responsible for the stimulation of skeleto-
genesis in corals by photosynthesis of zooxanthellae are not clear-
ly understood. If the two reactions are linked through some common
pathway, the coupling must be of a facultative type since cal-
cification can proceed in the absence of photosynthesis, although
at a much reduced rate. We have observed that calcification is
speeded up very quickly following the exposure of the corals
to adequate light intensities. The short time constant of the
potentiation makes it unlikely that the stimulation is due to produc-
tion of nutrients by the zooxanthellae, but rather to prompt changes

T. F. GOREAU 273

in concentration of some substrate common to photosynthesis and
calcification. In previous papers (3, 4) we advanced the working
hypothesis that acceleration of CaCOg deposition would occur if
algal photosynthesis were to remo\e COo from the system and cause
the equilibrium reaction

T
Ca(HCO,)., ^ CaCO, + H,CO,

to go to the right. Although the evidence for this is fairly pursua-
sive, other mechanisms may also be involved. Some of these will
be discussed below.

In principle, the rate of CaCO.; production could be stimulat-
ed in at least two ways : directly through control of the steady state
bicarbonate concentration in the tissues as shown above, or indi-
rectly by augmenting the supply of free energy available for active
calcium transport through an increase in the rate and efficiency of
cellular metabolism. In the discussion below, we will consider some
of the possible indirect mechanisms. The onset of photosynthesis
by the zooxanthellae immediately produces a rise in the intracel-
lular oxygen concentration which may result in some increase in
the rate and efficiency of metabolism in the coral. Thiel (15)
and Yonge (19), among others, have already emphasized the
probable importance of in situ production to the coral, but no spe-
cific mechanisms were proposed. There is at present no information
on the relation between the pO^. of the medium and the rate of
coral growth. Nearly all hermatypic corals are net oxygen producers
during the day, and the water circulating in the growing parts of
the reef is as a rule supersaturated with oxygen (8, 10, 11, 12, 13)
so that the dependence of calcification on oxygen would be diffi-
cult to measure in these organisms. In two ahermatypic corals
lacking zooxanthellae {Tuhastrca and Asirangia) we observed no
significant changes in calcium deposition rates under conditions
where the oxygen saturation of the medium varied between fifty
and one hundred and twenty two per cent, suggesting that calcifica-
tion rates in these corals are relatively independent of oxygen con-
centration within the limits tested.

274 THE BIOLOGY OF HYDRA : 1961

Given an adequate supply of oxygen in the medium, far reach-
ing effects on the rate and efficiency of metaboHc reactions can be
brought about by increasing the rate with which soluble waste
products are removed from the coral cells (20). This is a far more
potent metabolic stimulant than increasing the oxygen concentra-
tion. It has long been known that velocities of metabolic reac-
tions are strictly limited by the rates with which the end products
are removed from the immediate environment. In higher animals,
this is accomplished by specialised circulatory and excre-
tory systems which are lacking in the coelenterates. In the ab-
sence of zooxanthellae, or in darkness, corals are forced to rely
on diffusion alone to get rid of the soluble inorganic waste products
of cell metabolism. This is a slow process, especially when the sur-
face area for exchange is reduced by retraction of the polyps into
the calyces. This situation is radically altered in the presence
of zooxanthellae which require for photosynthesis and j)riniary pro-
duction those very substances that the coral host must get rid of,
e.g. COo, phosphates, nitrates, sulphates, ammonia, etc. Yonge and
Nicholls (21) showed for some corals that zooxanthellae are capable
of sufficiently high rates of photosynthesis to utilise not only all the
soluble inorganic phosphate produced by coral colonies, but that
additional phosphate is absorbed from the surrounding sea water.

Under conditions of adequate illumination, the zooxanthellae
are to be regarded as combined intracellular lungs and kidneys.
The observed speeding up of calcification in reef corals exposed to
bright light may in part be due to an increase of the rate and effici-
ency with which metabolism can supply free energy to the car-
rier mechanism concerned with active calcium transport. The ques-
tion whether the calcification rate is indeed related to the metabolic
rate, and whether this is in turn influenced by the level of algal
photosynthesis in the manner indicated above is now under
investigation in our laboratory.

CARBONATE DEPOSITION, GROWTH
AND PRODUCTIVITY

Elsewhere, we advocated the view that Ca~^ and HCO^g ions
dissolved in the ambient medium are the source of the mineral de-

T. F. GOREAU 275

posited ill the skeleton as CaCO^, and that these are brought to
the calcification site by separate pathways ( 3 ) . In order to test this
directly we developed a technique in which the uptakes of Ca"*'
and C^^ carbonate were measured simultaneously in a variety of
calcareous coelenterates and algae, under natural conditions in the
reef. As before, light and dark runs were carried out simultane-
ously, the experiments lasting lietween five and six hours. After
washing and drying the specimens, activities due to Ca^' and C^^
deposited in the skeleton were quantitatively isolated, and sep-
arated from the C^^ activity fixed in the coenosarc as organic mat-
ter by photosynthesis of the zooxanthellae. A detailed description
of this technique will be published later.

The data in Table I summarises results of field experiments
carried out for the purpose of measuring simultaneously calcium
and carbonate transfer rates from the medium into the test organ-
isms. The plants and animals used in these investigations, and list-
ed in Table I, belong to three different ecological categories: Group
1 consists of shallow water ahermatypic coelenterates which contri-
bute only insignificant amounts of calcareous matter to the reef;
Group 2 contains three hermatypic coelenterates which are chiefly
reef framework builders; Group 3 has three hermatypic algae, the
remains of which form the bulk of the fine calcareous lagoon and
slope sediments. All these species are found in the actively grow-
ing part of the reef rampart at Maiden Cay, Jamaica, where these
experiments were carried out.

The first two columns of Table I give the transfer rates of Ca"*^"^"*"
and HC^^O^g into the mineral skeleton, the third column gives
the rate of photosynthetic fixation of C^'' into organic matter, e.g.
the primary producti\'ity. In the ahermatypic coelenterates lacking
zooxanthellae, there are no significant light-dark differences in the
calcium deposition rates, but in the hermatypic coelenterates con-
taining zooxanthellae and in the hermatypic algae, these differ-
ences are extremely pronounced. An exception was the red alga
Ampliiroa where the calcification rate in darkness was much higher
than in light. Not unexpectedly, the organic carbon fixation val-
ues observed in ahermatypic species were extremely low, and were
probably due to heterotrophic exchange, or photosynthesis of bor-
ing algae in the skeleton.

276

THE BIOLOGY OF HYDRA : 1961

The primary carbon fixation observed in hermatypic coelenter-
ates was due to photosynthesis l3y zooxanthellae. The boring algae
were present in only very small amoimts in our samples and it is
assumed that their contribution to the total productivity was also
very small. Owing to uncertainty of the proportion of the plant
biomass in corals, the data are given in terms of total nitrogen, e. g.
animal plus plant. The highest calcification and productivity rates
were observed in the hermatypic algae. The two species of HaUineda
behaved like the hermatypic corals in that calcification was much
faster in light than in darkness, but in Amphiroa there was a nega-
tive correlation between photosynthesis and skeletogensis. We be-
lieve that light inhibition of calcification in this species is pro-
duced by a shortage of available carbonate due to competition for
CO2 as a common substrate by extremely high levels of photosyn-
thesis. This problem is now being investigated in our laboratory.

There is a positive correlation between the calcium deposition
rate and the photosynthetic rate as measured by the specific pri-
mary productivity, e. g. the amount of organic matter produced in

TABLE 1
Specific calcification and productivity rates of hermatypic and ahermatypic organisms.

Light

Category

Species

Calcium deposition
in j(i,g./mg.N/dav

Carbon fixation
in ;Ug/mg.N/day

Ahermatypic Coelenterata
without zooxanthellae

S. roseus

A. solitarid

T. tenuilamellosa

292.4
207.6
133.2

Hermatypic Coelenterata
with zooxanthellae

A. cervicornis
(apical cm.)
M. complanata
P. furcata

1936.8

1015.2
387.6

145.08

236.16
165.60

Hermatypic algae

H. tuna
H. opuntia
A. fragilissinia

3070.8

3944.4

10330.8

316.70
606.24
675.84

278 THE BIOLOGY OF HYDRA : 1961

played by photosynthesis in facihtating the deposition of calcare-
ous matter in a wide variety of hermatypic organisms, irrespective
of the possibility that the mechanisms concerned may be very
different.

Comparison of the results summarised in the first two columns
of Table I shows that skeletogenesis rates calculated from Ca^'
uptake are much higher than those calculated from the simul-
taneous C^^ carbonate uptake. In CaCO... the stoichiometric mass
ratio of calcium to carbon is 40 12 or about 3.335. This ratio should
apply to the mineral constituent of the coelenterate and algal skel-
etons which is mostly CaCO:., though some of the algae may con-
tain traces of dolomite in addition to calcite and aragonite (18).
However, the ratios calculated from our data are nearly all higher
than the theoretical value, and they vary over a wide range. This
either indicates that the organisms are secreting a skeletal mineral
greatly enriched in calcium, or that the specific activities of the C^^
and Ca^*" labelled percursors change with respect to the external
medium, and to each other, during the process of deposition. As
there is no experimental evidence for calcium enrichment we are
inclined to explain the apparent carbonate deficit shown in our
data on the basis of the second alternative.

The transfer rates given in Table I were calculated on the as-
sumption that during CaCO;, deposition the specific activities of
the Ca*'' and C^^ labelled percursors do not change with respect
to the sea water or to each other, a condition that would occur only
if the system were in isotopic equilibrium. However, this was not the
case in our experiments which were run over sufficiently short
periods of time that it was impossible for the test colonies to achieve
isotopic equilibrium. Therefore it is to be expected that the specific
activities in the newly formed skeletal CaCO;. would be less than in
the dissolved Ca+^ and HCO^ of the medium if the labelled ex-
ogenous atoms were to exchange with intracellular stores of un-
labelled atoms to final deposition into the skeleton.

Given that the molar fluxes of calcium and carl:>onate are equal
and linked by some common pathway, and using the specific activi-
ties of the precursors dissolved in the sea water as a reference
base, the calculated deposition rates will be the higher for that
component which has suffered the least isotopic dilution, e. g. cal-

T. F. GOREAU 279

cium, and the lower for that constituent which was diluted the
most during its passage through the cells, e. g. carbonate. This sug-
gests that the reservoir of intracellular carbonate available for ex-
change with absorbed exogenous carbonate is much greater than
the internal pool of freely exchangeable calcium, and that the tissue
calcium turnover rates must therefore be much higher than those
of carbonate. In previous experiments, we have demonstrated that
the exchangeable calcium in corals is indeed maintained at a low
level in corals (5, 7 ) . The simultaneous introduction of isotopically
labelled calcium and carbon makes it possible to assess the relative
sizes of the pools of exchangeable endogenous calcium and carbon
by the principle of dilution volumes in a situation where no isotopic
equilibration has occurred. Under these conditions, our calculated
transfer rates indicate that the internal pool of carbon available for
exchange with exogenous carbonate being deposited into the skel-
eton is about two to fifteen times greater than the amount of ex-
changeable calcium.

SUMMARY AND CONCLUSIONS

1. Coral reefs are tropical shallow water communities where
intensive biological calcification occurs, resulting in net accumula-
tion of limestone into the sediments. Photosynthesis appears to
be in some way essential to reef formation. The most important
reef-building organisms are calcareous algae and coelenterates, cor-
als included. All reef-building coelenterates without exception con-
tain symbiotic zooxanthellae. Corals without zooxanthellae grow
slowly and never play a significant role in the building of reefs.

2. The zooxanthellae do not themselves calcify, but their
presence results in a very powerful enhancement of calcification
in the coral host as soon as photosynthesis begins. We have shown
that stimulation of growth by light requires zooxanthellae since this
efi^ect does not occur in reef corals from which zooxanthellae are
removed, nor does it occur in ahermatypic corals which never have
algal symbionts. Of three calcareous algae tested, two calcified
much faster in light than in darkness, and in one the efl^ect was re-
versed.

280 THE BIOLOGY OF HYDRA : 1961

3. There is a rough correlation between calcification rate and
specific photosynthetic rate as measured by the organic productivity.
The highest calcification and productivity rates were noted in the
calcareous algae, but in one of these we observed a very strong
reduction of CaCO.j deposition in the light in the presence of a
very high rate of photosynthesis. Calcification and primary pro-
ductivity rates in three hermatypic coelenterates with zooxanthel-
lae are on the average about sixty per cent lower than in the
calcareous algae. Their slowest calcification rates were observed in
the ahermatypic corals that have no zooxanthellae.

4. Under the conditions of our experiments, it was found that
labelled calcium was deposited up to seventeen times faster than
labelled carbonate. This discrepancy may be the result of very
large diflFerences in the amount of exchangeable endogenous car-
bon in relation to the amount of calcium available for exchange,
the former being very much larger than the latter so that intracel-
lular dilution of the absorbed C^^ was much greater than that of Ca"* '.

5. Several mechanisms linking photosynthesis and calcifica-
tion are discussed. CaCOy production may be enhanced: (1)
through removal of CO2 from the calcification site by photosyn-
thesis and/or carbonic anhydrase; (2) from stimulation of coral
metabolism by photosynthesis of the zooxanthellae, which in turn
increases the amount of energy available for active calcium and
carbonate transport through the tissues into the skeleton. There
is no evidence that metabolic efficiency in reef corals is increased
by augmenting the oxygen supply over and above that already
available from the environment. The zooxanthellae probably exert
their effect by speeding up the rate with which metabolic waste
products are removed from the vicinity of the host's cells since the
algae require as raw material for photosynthesis those very inor-
ganic substances that the coral must get rid of. Rapid removal of
these from the host cells must set up strong local concentration
gradients resulting in a large increase of metabolic efficiency, thus
making more free energy available for a CaCO.^ secretion.

6. Photosynthesis plays a double role vis a vis the reef: it in-
creases the free energy of the community through primary produc-
tion and it produces in corals and algae the optimum physiological
conditions necessary for rapid and efficient secretion of calcium car-

T. F. GOREAU 281

bonate. In corals, the coupling of the calcification reaction to
photosynthesis, though facultatixe, is almost certainly due to a
direct link \'ia a common metabolic pathway, rather than to
synthesis and diffusion of nutrients from the zooxanthellae to the
host. There can be no question that the great increase in rate and
efficiency of limestone secretion associated with photosynthesis
must, on a community level, be of decisive importance to the for-
mation, growth and maintenance of tropical coral reef ecosystems.

REFERENCES

1. Emery, K. O., J. 1. Tracey and H. S. Ladd. 1954. Geology of Bikini and nearby

atolls. U.S. Gcol. Survey Prof. Pap. 260-A 265pp.

2. GoHAR, H. A. F. 1940. Stndies on the .Xeniidae of the Red Sea. Mar. Biol. Sta.

Ghardaqa, Egypt, Pub. 2: 25-118.

.3. GoREAU, T. F. 1959. The physiology of skeleton formation in corals. 1. A method
for measnring the rate of caleiimi deposition by corals under different
conditions. Biol. Bull. 116: 59-75.

4. GoREAU, T. F. 1961. Problems of growth and calcium deposition in reef corals.

Endeavour 20: 32-39.

5. GoREAU, T. F. and \'. T. Bowen. 1955. Calcium uptake by a coral. Science 122:

1188-1189.

6. Goreau, T. F. and N. I. Goreau. 1959. The physiology of skeleton formation

in corals. 11. Calcium deposition by hermatypic corals under various con-
ditions in the reef. Biol. Bull. 117: 239-250.

7. Gore.\u, T. F. and N. I. Goreau. 1960. The physiology of skeleton foniiation

in corals. IV. On isotopic equilibrium exchange of calcium between coral-
lum and environment in living and dead reef-building corals. Biol. Bull.
119: 416-427.

8. KoHN, A. J. and P. Helfrich. 1957. Primary organic productivity of a Hawa-

iian coral reef. Limn, and Oceanogr. 2: 241-251.

9. Ladd, H. S., E. Ingebson, R. C. Townsend, R. C. Russell and H. K. Stephen-

son. 1953. Drilling on Euiwetok Atoll, Marshall Islands. Am. Assoc. Petr.
Geol. Bull. 37: 2257.

10. Odum, H. T. and E. P. Odum. 1955. Trophic structure and productivity of a

windward coral reef community on Eniwetok Atoll. Ecol. Monogr. 25:
291-320.

11. Odum, H. T., P. R. Burkholder, and J. A. Rivero. 1959. Measurements of

producti\ity of turtle grass flats, reefs, and the Bahia Fosforescente of
Southern Puerto Rico. Inst. Mar. Sci. (Texas). Publ. 6: 159.

12. Sargent, M. C. and T. S. Austin. 1949. Organic productivity of an atoll. Amer.

Geophys. Union Trans. 30: 245-249.

13. Sargent, M. C. and T. S. Austin. 1954. Biologic economy of coral reefs. U. S.

Gcol. Surv. Prof. Pap. 260-E: 293-300.

282 THE BIOLOGY OF HYDRA : 1961

14. Teichert, C. 1958. Cold and deep water coral banks. Am. Assoc. Petr. Gcol.

Bull. 42: 1064.

15. Thiel, M. E. 1929. Zur Fra,^e der Ernahning der Steinkorallen und der Bredeu-

tung Ihrer Zooxanthellen. Zoo/. Anz. 81: 295.

16. Tracey, J. I., H. S. Lauu and J. E. Hokfmeister. 1948. Reefs of Bikini, Mar-

shall Islands. Geol. Soc Am. Bull. 59: 861-878.

17. Vaughan, T. W. and J. W. Wells. 1943. Revision of the suborders, families

and genera of the Scleractinia. Geol. Soc. Amer. Spec. Pap. 44: 363 pp.

18. Vinogradov, A. P. 1953. The elementary chemical composition of marine

organisms. Sears Found. Mar. Res. Mem. II: 647 pp.

19. Yonge, C. M. 1940. The biology of reef building corals. Gt. Barrier Reef Expecl.

Set. Rep. 7(13): 353-391.'

20. Yonge, C. M. 1957. Symbiosis. Geol. Soc. Amer. Mem. 67 (l): 429-442.

21. Yonge, C. M. and A. G. Nicholls. 1931. Studies on the Physiology of corals.

V. The effect of starvation in light and darkness on the relationship
between corals and zooxanthellae. Gt. Barrier Reef Expecl. Sci. Rep.
1(7): 177-211.

DISCUSSION

WAINWRIGHT: First I'd like to wave a small flag because you
who have trays of hydra in your laboratory and even you ocean-
ographers with laboratories in a ship don't have any idea under
what difficulties Dr. Goreau is working and what he has done in
taking his laboratory down onto the reef. Think of diving to 100 feet
with 200 pounds of machinery on your back and then doing a critical
experiment using glassware, radioisotopes, and lixing animals.

Now I want to ask a question. Do you know what the limiting
factors in calcification are?

GOREAU: No, not yet, if we exclude light for the moment. Con-
trary to what I said earlier, it may be possible to culture some species
of corals in vitro. We must never assume, however, that the growth
or accretion rates we measure under those conditions are equal to
those occurring on the reef. Nevertheless, laboratory studies are use-
ful because we can rigidly control the environment, the concentra-
tion of such substances as HCO^ and Ca++ and the additions of
inhibitors or stimulants, etc. We are planning such studies, but
haven't gotten around to them yet, so I cannot really answer your
question.

MUSCATINE: Do you feel that calcification in corals is augment-
ed by removal of COo by zooxanthellae?

T. F. GOREAU 283

GOREAU: Yes. If we assume the hypothetical scheme of calcifica-
tion which I published some years ago, then the removal of CO-
from the system would tend to drive the equilibrium to the right and
increase the rate of CaCO.; formation.

MUSCATINE: This differs from the scheme of Wilbur and Jod-
rey who found that calcification in their oyster mantle preparations
was increased about five fold if a source of COo such as oxaloace-
tate was added to the external medium.

GOREAU: Oxaloacetate is an intermediate in the Krebs cycle. Any
increase in the rate of this cycle may have rather non-specific effects,
and changes in calcification rates would tell us little. Nevertheless,
it's a very interesting possibility and we are planning work along
similar lines. Unfortunately, as Wainwright mentioned, there are
certain small difficulties in running such experiments.

MARTIN: In mammals, the accretion of bone substance is not a
one-way affair, but as accretion goes on, elimination and dissolu-
tion of bone material also goes on. I wonder if these views contri-
bute any insight into the problem.

GOREAU: Yes. This is a very important point. Bone and coral
differ in at least one fundamental way. Bone is mesodermal and
remains at all times part of the internal medium of the body. At
least 2(y/c of the bone mineral is exchangeable with calcium and
phosphate dissolved in the body fluids. In addition, mammalian bone
is vascularized and full of cells. The corallum, on the other hand, is
an ectodermal mineral deposit which lies outside the body of the
coral polyp. We have evidence that once the CaCO;^ is deposited
there, it undergoes little or no further exchange with the environ-
ment or with the coral; that is, it seems to be essentially isolated as
long as it is covered by a layer of living tissue.

LOO MIS: Dr. Goreau has shown that the rate of calcification at
the end of a coral branch is something like tenfold what it is at a
shoulder. I find this position effect fascinating since the two en-
vironments appear identical at first glance.

Another point is that CO^. plays a double role: (a) it is part of
the calcium carbonate which is part of the corallum, and (b) it

284 THE BIOLOGY OF HYDRA : 1961

exerts a pH effect. Now wlien the light is shining on the algae, free
CO2 is rapidly photosynthesized and the pH goes up to maybe 11
or 12.

GOREAU: Corals have alkaline phosphatases with optima at
about pH 11.0 (Goreau, 1953, P.N.A.S. 39: 1291). We thought
at first that these enzymes were concerned with calcifica-
tion, but results of our histochemical studies (Goreau, 1956. Na-
ture 177: 1029) make this appear unlikely.

LOOMIS: Under illuminated conditions you get precipitation
of calcium carbonate through increase of pH. Therefore, COo has
two roles in calcification: one as the carbonate ion, and one as free
CO2.

GOREAU: I am not sure that I agree with you. I wish we could
measure CO2 and pH in living calcifying corals. Let me comment on
the first part of your question regarding differential growth at tips
and sides of branches in Acropora cewicornis. Actually conditions
are almost certainly not identical at the tips and sides of branches.
This species has an inborn factor which controls the rate and pat-
tern of calcification in the colony — and thus determines colony
shape. It is a function inherent in the coral not the zooxanthellae,
and within some limits seems to have little relationship to photo-
synthetic carbon fixation as I mentioned in my talk.

PHILLIPS: How long a period of photosynthesis do you allow
in these experiments?

GOREAU: Approximately 6 hours,

PHILLIPS: Bean and Hassid (Assimilation of C^^Oo by a Photo-
synthesizing Red Alga, Iridophycus flaccidum. Bean, R. C. and
W. Z. Hassid. 1955. /. Biol Chem. 2i2;411-425) found in their
studies an assimilation of C^Oo in Iridophycus flaccidum, a ma-
rine red algae, that 90 odd percent of the C^^ was in an alcohol-
soluble phase. Alcohol extraction might be a possible way of
getting around your wet ashing. What is the method you use?

GOREAU : It is a modification of a technic published by Folch
and Van Slyke. Instead of using a mixture of concentrated sul-

T. F. GOREAU 285

phuric and phosphoric acids as the primary ashing agent, we use
mixtures of 70% perchloric and concentrated nitric acids with a
bit of potassium iodate added. We cannot use sulphate in any form
because we wish to avoid converting the calcium to the sulfate
and phosphate salts.

PHILLIPS: The 80% ethanol might be worth trying since it would
avoid the use of this rather explosive reaction mixture.

GOREAU: We have had no trouble with it because we are
using only 300 mg. samples in which there is less than 20 mgs. of
organic matter present.

HAND: Would you comment on the number of algae in the
growing tip as compared with the number farther away.

GOREAU: Histological sections show fewer zooxanthellae in
the growing tip of A. cervicornis. The mg. N/mg. chlorophyll a ratio
is also much higher in the axial polyps than in the lateral polyps-
indicating a lower specific photosynthetic rate in the growing tip.

HAND: This suggests that where there is less algae, there is
more calcification.

GOREAU: Yes, at least in A. palmofa and A. cervicornis.

The Development of Cordylophora^

Chandler Fulton

Department of Biolop.ij, Brandeis University, Waltham 54, Massachusetts

One of the challenging problems of development is the manner
in which a multi-cellular organism acquires and regulates its shape,
pattern, or proportion. Colonial hydroids offer especially favorable
material for study of this problem because their colonies are com-
posed of a repeating pattern of hydranths arranged on tubular
stems and stolons ( Fig. 1 ) . Hydroid colonies grow asexually by the
elaboration of stolons attached to a substratum; at regular intervals
the stolons send up uprights which bear hydranths, grow, and
branch. The primary concern of this paper is the manner in which
colonies develop this regular, repeating pattern.

I chose to work with the brackish-water hydroid, Cordijlophoia
lacustris, because it is exceptionally hardy and has a simple colony
pattern. For study of the development of colonies, it is advantageous
to have a refined and reproducible method of laboratory cultiva-
tion similar to that de\eloped by Loomis for Hydra Uttoralis. One
can grow Cordijlophoia colonies on glass microscope slides slanted
in beakers of culture solution, with no flow of water or other spe-
cial treatment ( 1 ) . The defined culture solution contains ionic so-
dium, potassium, calcium, magnesium, chloride, and bicarbonate.
All of these ions, with the exception of bicarbonate, are essential
for growth at a maximum rate, and the proportion of the ions is
critical. The cultures are fed Artemia larvae once daily, and the cul-
ture solution changed after feeding and again later in the day. Be-
tween feedings, the beakers are kept in the dark at 22 , though

lA much abridged form of the paper presented at the meeting. Relevant Hterature
citations and supporting data will be presented in papers to be published elsewhere,
and may be found in reference ( 2 ) .

287

288

THE BIOLOGY OF HYDRA : 1961

neither light nor shght variations in temperature are critical. These
standard conditions ( 1 ) have been used for all the experiments dis-
cussed here, since variation of the conditions leads to alterations in
colony pattern.

The number of hydranths in a Cordylophora colony increases
exponentially with time in the beaker-slide cultures, as do the hy-
dranths of Ilydia in Loomis cidtures. It is thus possible to compute
the growth rate of this colonial organism, using standard equa-
tions for exponential growth. This growth rate has been used to eval-
uate the growth conditions described above. Cordylophora colonies
double about every three days, or more slowly than Hydra littoralis,
which doubles in less than two days. The fact that Cordylophora
colonies grow exponentially even though they are colonial is ol
interest and we shall return to it later.

stolon lip

Figure 1. Diagram illustrating the basic pattern and macroscopic features
of a Cordylophora colony. Sketched from a photograph of a laboratory colony.

This culture method provided uniform Cordylophora colonies
with which I could begin to study colony fomiation. Time-lapse
movies taken to study the growth of colonies revealed a markedly
organized system of peristaltic waves, which probably act to circu-
late nutrients through the colonies.- These waves are proximally
oriented, beginning at the tip of each hydranth and passing down

-A movie demonstrating the features of peristalsis in Cordylophora was shown at tlie
meeting. The apparent synchrony of peristalsis is still being studied.

CHANDLER FULTON 289

through the tissue of the colony to the tips of the stolons. The waves
are rhythmic, though very slow, occurring about two or three times
an hour in a resting colony. The rate of peristalsis jumps threefold
on feeding, to a frequency of about eight times an hour, and
then declines back to the resting rate.

The most striking feature of this peristalsis is that it is sychro-
nized throughout a colony, in that the waves begin at the tip of each
hydranth simultaneously. Further, if one ties a ligature on any of the
uprights in a colony, the hydranth at the apex of that upright will,
in time, begin to beat out of synchrony with the rest of the colony.
In other words, disrupting the integrity of the colony (both tissue
and coelenteron fluid ) eliminates the synchrony. Even if one accepts
the conclusion that Cordylophora has nerve cells (Mackie, this
symposium), I find it difficult to envision how a stimulus is trans-
ferred through a colony in such a manner that each hydranth begins
a perstaltic wave at the same time. I would suggest, however, that
the synchrony indicates an order of integration in these colonial
organisms which we have not hitherto suspected. I suspect also that
understanding of colony development will involve further consider-
ation of the orientation, rhythmicity and synchronization of the
peristalisis.

On superficial examination, a Cordylophora colony looks like
a forest of little trees. I have attempted to distinguish the component
events which produce this forest, and in so doing have found it pos-
sible to describe in simple, quantitative terms how the forest de-
velops. Careful observation of colonies reveals that they are entirely
composed of a series of interconnected pipes, each consisting of a
cylinder of tissue surrounded by a tubular perisarc." These tubes
are of essentially uniform diameter. Thus one can conceive of a
Cordylophora colony as a plumbing system with 0.2 mm. pipelines;
the description of a colony can be reduced to a description of the
kinds of tubes which comprise it, the relative positions of these
tubes with respect to one another, and the way in which they are
formed and grow.

Stolon tubes, as they grow along the substratum, can give rise

^This approach to tlie colonies excludes the hydranths from consideration. Interesting
observations on factors influencing the shape of hydranths, as well as entire colonies,
have been presented by Kinne ( 3 ) .

290 THE BIOLOGY OF HYDRA : 1961

to two types of tubes: secondary stolons and uprights. Secondary
stolons leave their parent stolons at right angles along the sub-
stratum, while uprights leave at right angles away from the sub-
stratum. Uprights, in contrast to stolons, are hydranth-bearing
tubes, and give rise to one additional hydranth-bearing tube, the
side branch. Side branches leave upright tubes at about 45 degree
angles away from the substratum. Thus one can classify three types
of tubes: stolon, upright, and side branch.

Other differences further distinguish these tubes. Hydranth-
bearing tubes develop only directly behind growing tips; they never
develop in any other part of the colony. They are spaced at regular
intervals along their tube of origin; upright tubes in particular
occur at about three mm. distances along the stolon. In contrast,
stolon tubes never develop at growing tips, but always come out of
some old part of the colony, as at the base of a well-developed up-
right. Further, stolon tubes are not spaced regularly; rather secon-
dary stolons develop erratically with respect to any other part of the
colony.

How do these tubes grow? Since they are of uniform diameter,
one can detemiine the growth rate of individual tubes by measuring
increase in length with time. This has been done by photographing
a colony over the course of a few days or a week in a growth cham-
ber in front of a time-lapse camera. The movie is then used to plot
the extension of the tube as a function of time. Such plots, for both
stolons and uprights (side branch growth has not been measured),
demonstrate that these tubes increase in length linearly with
time. Stolons grow at a rate of about 0.1 mm. per hour, and uprights
at a rate of 0.05 mm. per hour.

You will recall that a colony as a whole grows exponentially in
terms of hydranth number. The colony also grows exponentially in
temis of dry weight, so that hydranth number is a measure of the
mass of a colony. The observation of linear growth of tubes poses
a dilemma : if the tubes which comprise a colony grow at a constant
rate how does the colony as a whole grow exponentially? This ques-
tion was first examined by model-building. One can diagram a col-
ony in the form of a geometric progression, such that linear growth
of tubes with regular branching at constant inten'als gives rise to
exponential growth of the whole. Such a model does not look

CHANDLER FULTON

291

like a Cordyloplwra colony in that 1 ) there is more branching than
in an actual colony, and 2) the uprights are too tall relative to
their parent stolons.

The geometric progression model was redrawn in terms of the
appearance of colonies growing under standard conditions, as
shown in Figure 2. The growth during any unit of time is indicated
by a pattern: black, stippled, etc. The stolon is visualized as grow-

r ^

i\\\\\\\t

Time units

Figure 2. A model illustrating the growth of a hypothetical colony over
a period of six time units. See text for explanation.

ing one unit per unit time (i.e., linearly), and producing uprights
at a rate of one per unit time. During the same time unit, an upright
grows only one-half unit, and a side branch only one-quarter unit.
However, uprights and side branches continue to produce new
tubes at the same distances as uprights are produced by stolons
(i.e., one unit), and thus produce new' tubes at rates of 0.5 and 0.25
tubes per unit time respectively.

Such a model takes into account the linear growth of tubes and
normal branching pattern, and gives rise to a two-dimensional
colony which bears a striking resemblance to laboratory colonies
(cf. Figs. 1 and 2), If one computes the increase in hydranth num-
ber of such a hypothetical colony with time, however, one finds that
it continually falls away from exponential. This is in contrast to act-

292 THE BIOLOGY OF HYDRA : 1961

iial colonies, which do approach exponential increase in hydranth
number. One can escape this new dilemma by doing what the colon-
ies do, namely by introducing secondary stolons at intervals. If one
adds such secondary stolons at appropriate times, one can make the
growth of the model colony closely approach exponential. I do not
know as yet whether or not this is the way colonies maintain ex-
ponential growth.

Laboratory colonies appear to develop in accord with the mod-
el. This has been determined by measuring every relevent variable
of the pattern of individual colonies, a task much facilitated by the
use of a marking technique. If colonies are dipped into trypan blue,
the perisarc is stained a deep blue while the tissue is unstained
and unaflFected. When such a colony is grown in the absence of
trypan blue, all new growth is colorless while that part of the colony
present as perisarc at the time of marking remains blue. Thus new
growth can be precisely measured as separate from old. The meas-
urements support the picture of colony formation just described,
except that branch tubes appear to grow more slowly, or at about
one-eighth the rate of stolon tubes. But upright tubes grow at al-
most exactly one-half the rate of stolon tubes.

In conclusion, it has been possible to reduce the development of a
Cordylophora colony to the growth and branching of a series of
tubes: stolons, uprights, and side branches. The parameters of col-
ony shape may be summarized in tabular form:

Source

Angle and position

Spacing

Relative

growth rate

Tube

Colonies Model

Stolon

Upright**

Branch**

Stolon
Stolon
Upright

90°, along substratum
90°, away from subst.
45°, away from subst.

erratic

r—^ 3 mm.
— 3 mm.

*These tubes also differ from stolons in that they bear hydranths and only develop
at growing tips.

A model has been developed integrating many of these aspects
of asexual colony development, and the development of indi-
vidual colonies studied in relation to the model.

CHANDLER FULTON 293

From my point of view, the major result of this study is that,
by reducing the development of a colony to a series of constituent
events, it becomes possible to analyze the individual events which
give rise to the shape of a colony. Many questions immediately pose
themselves. For example, why do upright tubes grow at half the rate
of stolon tubes? Why do hydranth-bearing tubes develop only be-
hind growing tips, while stolon tubes develop away from these tips?
What produces the regular spacing of upright tubes? What de-
termines the angle at which each tube leaves its parent tube? As
yet, none of these questions has even a preliminary answer, but I
hope that at least I have provided you with a more dynamic pic-
ture of these hydranth-bearing pipelines.

REFERENCES

1. Fulton, C. 1960. Culture of a colonial hydroid under controlled conditions.

Science 132: 473-474.

2. Fulton, C. 1960. The Biology of a Colonial Hydroid. Ph.D. Thesis, The Rocke-

feller Institute, New York.

3. KiNNE, O. 1958. Adaptation to salinity variations: some facts and problems. In

Physiological Adaptation ( C. L. Prosser, ed. ) . Washington, American
Physiological Society, pp. 92-106.

DISCUSSION

MACKIE: Before the discussion turns to the main topics of Dr.
Fulton's paper I'd like to comment on the colonial rhythm shown by
Cordylophora— the synchronized waves of peristalsis in the hy-
dranths. We have also seen this in Dr. Strehler's film of Fennaria.
This sort of activity demands a specialized conduction system. Re-
cently, R. K. Josephson has recorded action potentials from the
stems of Cordylophora and Tuhularia. I cannot give the full details
but in Tuhularia there are two rhythmically occurring patterns of
activity and one of these patterns has distinct motor effects.

I'd also like to reiterate that neurons have been identified his-
tologically throughout stems and hydranths in Cordylophora, so
there's no need for scepticism about the existence of a ner\ous
system in these colonial forms.

294 THE BIOLOGY OF HYDRA : 1961

CROWELL: This frequency (of three times an hour or so for hy-
dranth movement) surprises me, because, in the stolon anyway, if
one watches the movement back and forth of the fluid, one gets a
periodicity in the order of 3 to 5 minutes in all the hydroids I've
looked at.

FULTON: Have you looked at Cordylophora?

CROWELL: Yes.

FULTON: In the Cordylophora stolons I've followed, there are
a pair of filling and emptying cycles about every twenty minutes,
which corresponds to the frequency at the hydranths.

CROWELL: I don't doubt that. What you see in the hydranths,
I think, is different from the typical back and forth flow in the
stolons.

FULTON: I don't think so, but we're still in the process of finding
out.

CHAPMAN: I wonder if you have any information about tlie
relationship between culture conditions, such as tonicity, tempera-
ture, and pH, on the spacing of these uprights?

FULTON: I have voluminous information. Actually not much
affects interupright spacing, but many things affect the general
pattern of colonies. Kinne has made a thorough study of the ef-
fects of different dilutions of seawater and of different tempera-
tures on colony pattern. I think that all it would be wise to say right
now is that the pattern which I get is the pattern one gets in stand-
ard culture solutions at 22° with one feeding a day and all the ritual.
One can get almost any colony shape one wants simply by varying
one parameter or another. So this is quite a labile system.

LOOMIS: What strikes me in your nice growth records is a sort
of feeling that the stolon is trying to escape from itself. In other
words, it is trapped in its own one dimensional line and starts grow-
ing a shoot upwards. Then growth has to escape from this shoot
and does so first to the right and then to the left. New growth largely
takes place in a new axis at right angles to old growth, which is
another way of saying that growth can take place only at an open

CHANDLER FULTON 295

and advancing tip. This growth inhibition along an estabhshed
stolon may be related to the fact that Cordtjlophora, unlike hydra,
will not grow on the bottom of a Petri dish but needs to be suspend-
ed on a microscope slide in a beaker of water. The reason for this
"Fulton effect" as I call it, seems to be the greater sensitivity of
Cordylophora to pC02, for we have found that a pC02 as low as
1.5% atm. inhibits its growth while Hydra can stand up to 10% atm.
Thus, Cordylophora on the bottom of a Petri dish sits in its own
"halo zone" of high pCOo and inhibits itself, whereas Cordylo-
phora on a slide is continually bathed by the thermal currents that
exist within a beaker and can easily be shown with methylene blue.
Perhaps this apical inhibition of stolon growth by pCOi. may
partially explain the growth pattern of Cordylophora.

Developmental Problems
in Cam^anularid

Sears Crowell"

Departtiient of Zoology, Indiana University, Blooiningtun, Indiana

This report reviews a limited number of experimental studies
on the thecate or calyptoblastic colonial hydroid Campanularia
flexuosa (Hincks). The selection of topics has been biased by the
fact that most studies of developmental problems in hydroids have
employed either hydra or athecate (gymnoblastic) species (e.g.
Cordylophora, Corymorpha, Hydractinia, Tuhularia). This report
1 think, can be most useful if emphasis is placed upon the peculiar
features of thecate forms and on differences between the two groups.
No attempt has been made to cover comprehensively the morpho-
genesis of thecate hydroids or related work of other investiga-
tors. I have tried to point out a few of the interesting unsolved
problems

The principal topics are:

1. Patterns of colonial growth

2. Alterations of the pattern of growth

3. Aging

4. Regression and replacement of hydranths

5. Reconstitution studies

6. Hvdranth differentition

iThe research lias been supported by a research grant (H-1948) from the National
Heart Institute, U.S.P.H.S., and by a grant-in-aid from the American Cancer Society.

^Department of Zoology, Indiana University, Bloomington, Indiana and the Marine
Biological Laboratory, Woods Hole, Mass. Contribution No. 707 from the Depart-
ment of Zoology, Indiana University.

297

298

THE BIOLOGY OF HYDRA : 1961

PATTERNS OF COLONIAL GROWTH

This brief report cannot cover the extensive hterature on pat-
terns of growth. By 1914 Kiihn (10) had provided a comprehen-
sive review and his figures have been used and recopied ever since.
Recently Berrill has clarified many points concerning hydroid
morphogenesis, and his recent book (2) provides us with both an
excellent survey and a bibliography.

The pattern of colonial growth of a typical athecate hydroid is
shown in Figure 1 A. The oldest hydranth, terminal in position, is
designated as 1, the next oldest, 2, etc. There is a zone of growth
just proximal to each hydranth. Each such zone contributes to furth-
er increase in the size of the colony in two ways: it lengthens the
pedicel or stem in which it lies, and it gives off laterally at regular
intervals a new hydranth bud with its own distinct growth zone. A
newly produced hydranth initially has few tentacles and is small.
Tentacles are gradually added as the hydranth grows in size. It is
easy to determine the relative ages of the hydranths of a col-
ony with this growth pattern, on the basis of both the position and

7 /^6

2 3

Fig. 1. Diagrams to show the growth pattern of colonial hydroids. A. The
pattern typical of most colonial athecate species. B. The pattern of many
thecate species, e.g. Campanularia, Obelia. The black regions are zones of
growth. The numbers show relative age of hydranths. From Kiihn (10).

SEARS CROWELL

299

the size of hydranths. This is clearly illustrated in the photographs
of Cordijlophora (Fig. 2) and Pcnnaria (Fig. 3); and both corre-
spond almost perfectly with the idealized pattern of Figure 1 A.

Fig. 2. Pattern of a small colony of Cordylophora.
graph by Charles Wyttenbach.

From a color photo-

Figure 1 B illustrates the typical growth pattern found in many
thecate species. Growth zones give rise to the stems (pedicels) of
new hydranths but do not add to the length of the stem itself.
Hence the order of the age of the hydranths (in a young colony) is
from the base upward, 1, 2, etc., in Fig. 1 B. The youngest hy-
dranth is terminal— the opposite of the pattern in athecate species.

In thecate species the pedicel of a new hydranth is completely
formed before the hydranth itself is produced. After the pedicel at-
tains its full length it enlarges at its tip to make a hydranth bud,
which then quickly differentiates into a hydranth. By the time the
hydranth emerges from its enclosing hydrotheca, it is fully func-
tional, and has its full set of tentacles and its full size. It grows

300

THE BIOLOGY OF HYDRA : 1961

no more. The photograph of Campamdaria (Fig. 4) shows that all
hydranths are of the same size. The bud of the hydranth which will
be produced next is at the top, and proximal to this is the begin-
ning of the outgrowth of the next pedicel.

Some species of both thecate and athecate hydroids are solitary,
and there are other species in which all hydranths arise only
from the attaching stolon (e.g. Htjclr actinia). Yet another pattern
of colonial growth, in which the growth zones are apical, is seen
in sertularians and plumularians— presumably the most advanced
of the thecate hydroids. These too provide challenging problems
for experimental morphologists but cannot be considered here.

Fig. 3. Pattern of a small portion of a
colony of Pennaria. From a color photo-
graph by Charles Wyttenbach.

Fig. 4. (right). Pattern of growth for
Campanularia flexuosa. From a color photo-
graph by Charles Wyttenbach.

SEARS CROWELL 301

The precise patterns of growth in hydroids tempt one to con-
struct mathematical models such as those which Fulton has devel-
oped and presented so well in this symposium. I am confident that
similar models could be constructed for Campamdaria. The pre-
ciseness of patterns also invites us to attempt to alter them.

ALTERATIONS OF THE PATTERN OF GROWTH

The basis for our first studies on Campanularia was the belief
that procedures which would alter the pattern of growth would
give some insight into the underlying controlling conditions.
Young colonies grown at different temperatures gave colonies or
similar form, but their growth schedule was strikingly altered. At
higher temperatures the apical growth of each new pedicel and
hydranth was accelerated, l)ut at cooler temperatures the initiation
of the growth of each new pedicel occurred so much sooner that
these colonies as a whole grew just as rapidly (7).

This experiment showed that the factors which control the initi-
ation of new growth are different from those which control rate of
growth in an already established growing region.

In a more elaborate experiment all growth zones and prospec-
tive growth zones were compared in colonies kept at different nu-
tritional levels. Figure 5 C shows diagramatically all of these zones.
It could be predicted that with sub-optimal feeding there must be
either a general uniform slowing down of all activities or a favoring
of some at the expense of others. The latter proved to be the case.
In general, lowered nutrition did not greatly affect the rate of
growth in an already established part, but it did delay or stop the
initiation of new growth. For example, the main stolon grew
almost as well in nearly starved specimens as in well fed ones,
and it produced new uprights. However, the initiation of subterminal
growth by the uprights was delayed. As a consequence of these two
effects the whole pattern of partly starved colonies was strikingly
different from that of well fed ones. The two were about equally
extensive along the substrate, but the height was conspicuously
different.

302

THE BIOLOGY OF HYDRA : 1961

It is easy to conjecture that this difference is adaptive in na-
ture: It is better for a colony at an unsatisfactory feeding site to
move along than to add more feeding units where it is.

Fig. 5. Campanularia. A. Technique of subculturing by placing an up-
right beneath a thread which has been tied around a slide. The new growth
is suggested by the dotted line. B. Pattern of a colony of the age used in
the nutrition experiment discussed in the text. The numbers designating
age of the upright correspond with those in Fig. 6. C. The zones of growth
and prospective growth in Campanularia are indicated: W. to Z. (With
permission; Fig. 1 of ref. 4).

AGING

The most striking observation, by serendipity, in the experi-
ment just discussed was that the increase in height of the older
uprights (stems with their hydranths) was much more adversely
aflFected by reduced nutrition than the comparable growth of young-
er uprights in the same colony. The growth in length of the up-
rights depends on recurrent initiation of each new node— it is

SEARS CROWELL

303

intermittent, not continuous. Figure 6 summarizes the experimental
results. The groups are arranged in the order of decreasing food
supply, and in each group the oldest upright is No. 1 at the left. In
the well fed groups, glut and 4 2, old and young uprights had grown
at the same rate. In all the others the younger grew faster (4).
The effect of age of stem in slowing or limiting terminal growth
was studied further (8). In one test the more basal levels of an up-
right were removed every few days so that it consisted of only the
4 to 8 youngest hydranths. The terminal growth, in these cases,
did not stop; the total length of stem produced was more than three

12 3 4 S 6 7 8 9 10

u uuuuuuuuu

I 13 4 5 6 7 B 9 10 II

uuuuuuuuuuu

GLUT

0 —

<- I.

r~ini~ir~ir-ir-ir-n— inrnf— 1

JD.

2 5/

JIL

jnEL

dddD

r-ii-ir-n-ir-ir-n-ii-ir-n-n-i

34 56 78 9 10 II

Fig. 6. Terminal growth related to nutritive level and to age (height) of
upright. The subfigures are arranged in decreasing order of nutritive
level; within each the oldest upright is at the left. (With permission; Fig. 3
of ref. 4).

304 THE BIOLOGY OF HYDRA : 1961

times greater than that observed in normal specimens or than that
reported as the maximum height for this species in nature. Oth-
er experiments, but not all, showed evidence of an aging factor
inhibitory to growth. These studies are being continued.

REGRESSION AND REPLACEMENT OF HYDRANTHS

In all thecate hydroids which have been examined hydranths
are short-lived; they regress and are resorbed after about one week
(3). In Figure 4, for example, it may be noticed that there is only
a pedicel at the location, lowest left, where the oldest hydranth
"ought" to be; it had regressed. In this symposium. Dr. Strehler is
presenting much of our information ( 12 ) concerning this regres-
sion-replacement cycle and its implications for the understanding
of aging.

When regression occurs, the materials of the hydranth go back
into the colony and are available as nutrition for further growth,
a point which has been proved by Berrill ( 1 ) and Nathanson (11).
[See comment by Crowell in the discussion of the paper by Streh-
ler in this symposium (p. 396).]

In contrast with thecate species athecate hydroids do not re-
gress, so far as we know, except under adverse conditions. We have,
for example, records of Cordylophora hydranths which lived for
more than three months even when food was limited and growth
was almost at a standstill ( not previously reported ) .

RECONSTITUTION EXPERLMENTS

Hydroid tissues can be dissociated mechanically giving tiny
clumps of cells, which can be pushed together into a loose mass. In
both thecate and athecate species these clusters reorganize them-
selves into a double-layered hollow sphere with epidermal cells on
the outside, endodermal cells inside. Up to this point thecate and
athecate tissues are similar in behavior. The subsequent events
differ strikingly and emphasize in a different way the contrast be-
tween the two groups in the manner in which a hydranth develops.

SEARS CROWELL

303

In a day or two in Coidijloplwra and other athecate forms a small
bud (sometimes several) appears on the upper side of the cellular
ball and quickly develops four or so tentacles. If fed, it will grow.
In Campaniihha, a growth zone appears on the ball and produces
either a stolon or a pedicel. This grows out for several days using
the materials in the ball. Finally, after about a week, in exactly
the same sequence as in ordinary hydranth development, a new
small but complete hydranth is produced. Figure 7 shows sketches
of this for Campanularia.

Fig. 7. Sketches of the production of hydranths from dissociated tissues
of Campanularia. At the top are clumps of cells which have been pushed
together. Within a few hours these rearrange themselves into a hollow
ball. This ball may produce a hydranth in either of two ways: at the left,
by the production of a stolon from which a pedicel and then a hydranth
develops; at the right, by the production of a pedicel at the top of which
a hydranth develops (From Hartman, ref. 9).

306 THE BIOLOGY OF HYDRA : 1961

Here again we must raise the question: What is it that is being
moved from the old jDart to make the new? Are cells moving? Are
old cells breaking down to give substances that are moved and
reutilized? We do not yet know the answers.

At Indiana, Mr. Hartman (9) undertook to find differences
among the tissues taken from different parts of a colony in re-
spect to their capacities when dissociated. No differences were
found among tissues from stem, stolon, or early hydranth buds of
Campamdaria. Tissue taken from adult hydranths, however, did not
reconstitute. This led, naturally, to tests of different stages of hy-
dranth development. When a late stage of hydranth development
was used, but one in which there was not yet any visible differentia-
tion, Hartman found that the tissues reaggregated and within a
few hours produced differentiated hydranth parts with an ir-
regular organization. Two examples showing patches of tentacles,
and in one case a hypostome, are illustrated in Figure 8. Evidently

Fig. 8. Two examples of the irregular structures which differentiated
when tissues from a late hydranth bud were dissociated and allowed to re-
aggregate. There are patches of well developed tentacles, and in the
example at the right there is a hypostome (From Hartman, ref. 9).

each region of the scrambled tissues was already set in the course
of its differentiation. A further test of the distal tissues of buds at
this age showed that they were like the whole in making irregular
structures at once. But the tissues taken from the proximal halves
of such buds reconstituted according to the same pattern as stem,
stolon, or early bud tissue.

SEARS CROWELL

307

HYDRANTH DIFFERENTIATION^'

The manner of development of thecate hydranths, their failure
to grow, and the fact that they regress after only about a week sug-
gest that they have little regenerative or regulative ability. We have
cut tentacles from young hydranths and find that they do not
regenerate appreciably. If the hypostome is cut off regression en-
sues within a few hours. To carry this matter further back into
stages of hydranth development we undertook several types of sim-
ple operations on hydranth buds.

Athecate hydranths which have had parts removed replace
them. The three sketches of Figure 9, for example, illustrate the

-T-rrrfi-^-TTr

Fig. 9. Rapid restoration of tentacles and hypostome in Cordylophora
following the removal of the hypostome and most of the tentacles.

^The experiments described in this section have not been presented elsewhere except
in abstracts (5, 6 ) .

308

THE BIOLOGY OF HYDRA : 1961

quick regeneration which followed removal of the hypostome and
most of the tentacles in a small young hydranth of Cordylophora.
An analagous operation, illustrated in Figure 10, was performed
several times on hydranth buds of Campanularia. Both the excised
piece and the part which remained proceeded to differentiate just
as they would have if no operation had been made. The isolated lit-
tle pieces consisted of little more than tentacles and a hypostome.
Such little creatures captured Artcmia larvae and passed them into
the hypostome. They lived unchanged for about four days— a nor-
mal life span for an unnourished hydranth. Similarly the "half hy-
dranths" still on the colony showed normal activity but no restitu-
tion of the missing tentacles.

fh hr5

Fig. 10. The left half of the upper portion of a late hydranth bud of
Campanularia is cut off. Both parts differentiate just what they would have
produced normally, and there is no later restoration of missing parts.

In another series of experiments we cut off and isolated very
young hydranth buds of Campanularia, as shown in Figure 11. These
were of such small mass that it would be impossible for them to
develop a normal hydranth. Had these been athecate hydranth buds
one would have predicted that they would produce either nothing,
because of the small size, or at best a tiny hydranth. These isolated

SEARS CROWELL

309

Fig. 11. Profile sketches of the morphogenesis of an isolated early hy-
dranth bud of Campanularia. The finally differentiated disk consists of
little more than a hypostome surrounded by a full circle of tentacles. The
outer line represents the secreted perisarc; the tissue is stippled.

buds of Campamdaria, however, showed an extraordinary ability to
continue to perform the activities ordinarily performed by the
distal-most tissues of a normally developing hydranth. They gradu-
ally spread themselves laterally, laying down externally the hydro-
thecal perisarc, and they continued to do so until a hydrotheca of
ordinary size was produced. By this time the tissue itself was only
a thin disk at the position where hypostome and tentacles would
differentiate in a whole bud. Then the disk differentiated into just
these distal-most parts.

The whole process just described proceeded much more slow-
ly than is the case in normal development. If one were dealing
only or mainly with cell migration it would be expected that the
events could occur at nearly normal speed. The slowness suggests
that new cells are being produced, as is believed to be the case
in ordinary hydranth development, and old ones are being de-
stroyed and utilized. Regardless of the validity of this sugges-

310 THE BIOLOGY OF HYDRA : 1961

tion, it is clear that distal-most tissues have held rigidly to the se-
quence of events characteristic of these tissues in normal
development.

CONCLUSIONS

It is clear that the pattern of colonial growth can be altered in
Campanularia by changes both in temperature and in nutritive
level. The alterations are largely due to the sensitivity of zones
of prospective growth.

Differences in hydranth morphogenesis are striking when one
compares the processes in thecate and athecate species. In the
thecate form, Campanularia, a hydranth of full size is produced by a
series of events which are not easily altered; they show little abil-
ity to regulate. Once produced thecate hydranths do not grow,
they do not regenerate parts which have been removed, and they
regress and are resorbed after living for only a few days. In all
these respects the reverse is true for athecate species.

We know that old parts are utilized for new growth, but we do
not know in what form materials are moved: as tissues? cells?
fragments? chemical substances? This needs study. More attention
also should be given to the initiation of new growth by zones of
prospective growth. For analysis of these particular problems the-
cate species, such as Campanularia, are probably better than athe-
cate forms.

ACKNOWLEDGEMENTS

The author must acknowledge the assistance of Malcolm Rusk
and Ruth Curtiss Telfer who were with him at the beginning of
the studies of Campanularia; of Charles Wyttenbach who has made
many contributions of ideas and time and whose photographs have
been copied here; of Fred Wilt, Richard Manassa, Annelle Gibbon,
Jean Lowiy, Maurice Hartman, and Pat Clapp all of whom have
had some part in the work summarized here.

The paper ought to be dedicated to the memory of Frederick
S. Hammett who long ago proclaimed the special virtues of Cam-
panularia for studies of growth.

SEARS CROWELL 311

REFERENCES

1. Berrill, N. J. 1949. The polymorphic transformations of Ohelia. Quart. J. Micr.
Set. 90: 235-264.

2. Berrill, N. J. 1961. Growth, Development, and Pattern. W. H. Freeman and

Company, San Francisco. 555 pp.

3. Crowell, S. 1953. The regression-replacement cycle of hydranths of Obelia

and Campanularia. Physiol. Zool. 26: 319-327.

4. Crowell, S. 1957. Differential responses of growth zones to nutritive level, age,

and temperature in the colonial hydroid Campanularia. J. Exp. Zool. 134:
63-90.

5. Crowell, S. 1960. Non-regulative differentiation in the thecate hydroid Cam-

panularia. Anat. Rec. 138: 341-342.

6. Crowell, S., and M. Hartman. 1960. Reorganization capacities of dissociated

tissues of Campanularia flexuosa. Anat. Rec. 138: 342.

7. Crowell, S., and M. Rusk. 1950. Growth of Campanukiria colonies. Biol. Bull.

99: 357.

8. Crowell, S., and C. Wyttenbach. 1957. Factors affecting terminal growth in

the hydroid Campanularia. Biol. Bull. 113: 233-244.

9. Hartman, M. E. 1960. A study of tlie reorganization capacities of dissociated

tissues of Campanularia flexuosa. M.A. Thesis, Indiana University.

10. KuHN, A. 1914. Entwicklungsgeschichte und Verwandtschaftsbeziehungen der

Hydrozoen. I Teil: Die Hydroiden. Ergeb. Forschr. Zool. 4: 1-284.

11. Nathanson, D. L. 1955. The relationship of regenerative ability to the regres-

sion of hydranths of Campanularia. Biol. Bull. 109: 350.

12. Strehler, B. L., and S. Crowell. 1961. Studies on comparative physiology of

aging. I. Function vs. age of Campanularia flexuosa. Gerontologia 5: 1-8.

DISCUSSION

FULTON: I am much impressed with the similarity of the
growth pattern of Campanularia and Cordijlophora. For example,
if you starve Cordt/lophora, the stolon is the least affected part.

CROWELL: We didn't say anything about longevity. Cordijlop-
Jiora hydranths don't die after a week or so as do calyptoblast hy-
dranths.

FULTON: As far as I know Cordijlophora hydranths never die.

STREHLER: I would like to speak on that point. We have studied
Bouganvillia hydranths for as long as 25 days and haven't seen
a single individual die. They continued to increase in size as they
got older. On the other hand the oldest Campanularia that we've

312 THE BIOLOGY OF HYDRA : 1961

ever found is eleven days of age. That's at about 17°. You can
find older ones if you lower the temperature. Clijtia, by contrast to
Campanularia adjusts in size to the amount of food that is avail-
able. In Campanularia you get essentially the same size hydranths
regardless of how well or poorly one feeds the colony. If it starts
to make a hydranth it makes one of the standard size. Although
Clytia hydranths do vary in size they don't grow after they're fully
formed. You can get very tiny hydranths if the colony is starved
and some hydranths as large as Campanularia if they are well fed.
If Clytia is growing on Artemia and for some reason they don't
catch their food on a regular basis, they very soon get to a size
where they can't ingest Artemia because none of the hydranths are
large enough.

CROWELL: There is some variation in Campanularia. If one
uses tissue masses of different sizes, one finds that there is a lower
limit where one gets no hydranths. Above that, one gets specimens
somewhat smaller than normal and with a smaller tentacle number.
Then if one uses still larger masses one gets correspondingly
larger hydranths. It's not very striking though.

STREHLER: There is one implication in a word that you used.
You said that there was a zone of "proliferation" down near the
developing bud and I just wonder how you would explain certain
experiments we did last summer which consisted of giving a colony
100,000 r of X-rays, enough so that the slides on which they were
growing became deep amber in color. Still, after ten days, a few new
hydranths were formed in the radiated colony. Just a few, it's true.

CROWELL : Subterminal hydranths?

STREHLER: These were replacements, I believe, i.e. subterm-
inal. The point is, that it's hard for me to see how cell division
could occur after that amount of radiation. I would propose alter-
natively, that there are cells which have aheady divided and
which probably lie in the stolon. At the proper signal these cells
migrate into the region of what one might call growth, but which
I think may better be considered as regions of differentiation and
morphogenetic movement where no cell division is taking place.

CROWELL: I think what you suggest is x^erfectly possible. The

SEARS CROWELL 313

evidence for mitosis in these growing tips is most unsatisfactory.
Berrill says mitosis occurs in growing hydranths but he never pre-
sents any illustration of this mitotic activity. This is one reason why
Mr. Lunger is now trying to look at these growth zones using the
electron microscope. We hope to understand these processes at the
cellular level. We certainly cannot right now.

STREHLER: At the end of this afternoon's session I hope to
show some time-lapse movies of an irradiated colony. I call this
movie "On the Beach."

FULTON: Can I interject something? I have been trying very
hard to find out where cell division occurs in Cordylophora. I
don't know whether it's me or the animal, but I cannot see any
chromosomes. If anybody knows how to see mitosis in adult hy-
droids I would be very happy to hear of it.

CROWELL : Send me a copy of the letter.

LYTLE: The only place we have been able to find mitotic figures
in Cordylopliora is in early embryos.

FULTON: This is easy.

LYTLE: Not as easy as one might expect. We had to look at a lot
of sections to find any mitotic figures.

FULTON: Adult tissues must divide for they grow about one-
tenth of a millimeter an hour. There must be cell division some-
where.

SLAUTTERBACK: In reference to the transected bud, I was
quite interested in your "rob Peter to pay Paul" expression. I
take this to mean that any one cell possesses not just a single pat-
tern of differentiation, but all the possible patterns necessary for the
production of a whole hydranth. And in this case, a cell may car-
ry out each of these patterns sequentially until it has gone through
all the steps normally carried out by many different cells. Do
I understand correctly, or is there some mitosis going on and it is
the daughter cells which make tentacles where the parent cell has
made perisarc or stem or something else?

CROWELL : I don't think we know.

314 THE BIOLOGY OF HYDRA : 1961

SLAUTTERBACK: This intrigues me very much because we've
come upon dedifferentiation and redifferentiation in the pedal disc.
If one amputates the pedal disc, the secretory cells are soon re-
placed but not from the undifferentiated interstitial cell as might
be expected, but by partial dedifferentiation of cnidoblasts fol-
lowed by differentiation into secretory cells. This observation is
possible because the nematocysts persists in these cells throughout
the process. In fact, the mature secretory cells often contain a part-
ly disintegrated nematocyst. Furthermore, even the organelle devel-
opment characteristic of the cnidoblast persists for a time after the
secretory cell, with its very different organelles, has begun to func-
tion. I think this is one of the rarer demonstrations of a partial de-
differentiation and then redifferentiation of the same cell into an
entirely different cell line. I wonder if that is what is going on in
your situation, or whether you have mitosis intervening, or what?

CROW ELL: If one starts with a little colony consisting of a stolon
and a few hydranths, and does not feed it, one often finds that there
is new growth of the stolon and then production of new hydranths
from the new stolon. I have seen this in Campanularia and Cor-
dylophora; Berrill has described it. Of course, as new stolon and
hydranths are growing at one end, old hydranths and stolon are
regressing at the other end. One does get regression of hydranths
of Cordylophora in this situation; however, there is no regression
in well fed colonies. Of course, such a system gradually gets small-
er—as long as it lasts it produces new parts at the expenses of
the old.

SLAUTTERBACK: I wonder whether there is a degradation of
cells followed by reuse of the degraded material to make new cells,
or whether there is a dedifferentiation, migration and redifferentia-
tion of the original cells from the old hydranths.

CROW ELL: That is just the point that is not understood.

SLAUTTERBACK: In the pedal disk it is the old cells that are
reused, i.e. redifferentiated.

FULTON: This must also be the case in Cordylophora because
the stolons of a starving colony will keep extending over the slide
for months; the hydranths and stolon tissue behind the advancing

SEARS CROWELL 315

tips being resorbed and regenerating continuously. Since there is
no other source of nutrients, old cells must be reused. In line with
this, I wanted to emphasize that there is normally no regression of
hydranths in Cordylophora. Kinne ( 1956, Zool. Jahrb., Abt. Phy-
siol. 66: 565 ) followed individual hydranths for about 140 days, and
I have observed them for several months with no indications of
regression.

STREHLER: Does the hydranth continue to get larger during
all that time?

FULTON: They may grow very very slowly. They reach adult size
I'd say in about a week of growth.

CROWELL: You haven't said whether new cells are being pro-
duced by using substances derived from old cells, or whether the
same cells are producing the new parts by migrating.

FULTON: I don't know. All I'm saying is that they can't be
using up too much because they will go on for months.

CROWELL: It will go on a long time.

LYTLE: Or it's a very efficient system for recycling materials.

STREHLER: It would be very interesting to know whether the
same cells stay in the fully formed hydranth if it's not growing.
That is, is there a cycle of cell replacement? One should be able to
find out by seeing the effect of large doses of X-radiation on the
longevity of CorchjJophora. Will it kill them in the same doses
which double the longevity of Campanularia?

SLAUTTERBACK: Every attempt we have made to demonstrate
an increased mitotic rate following amputation of hydra heads
has been unsuccessful. The formation of a new head with its
tentacles appears to be strictly a matter of migration of cells from
the column. There is no change in the level of differentiation of
these cells nor is there any visible increase in mitotic activity.

CROWELL: How successful are you in finding mitosis down in
the lower region?

SLAUTTERBACK: We can see them fairly commonly in the in-

316 THE BIOLOGY OF HYDRA : 1961

terstitial cells with the electron microscope but not with the light
microscope.

LENHOFF: We measure changes in the number of nematocysts
in H. littoralis using a specific test for hydroxyproline, the imino acid
that makes up much of the nematocyst capsule. We find that de-
capitated Hydra which regenerate complete sets of tentacles show
no net increase in hydroxyproline although starved Hydra are able
to synthesize this unessential imino acid. Thus, it appears that re-
generating animals use the nematocysts that they already have in
their body tubes, and no new increase in the number of cnido-
blasts occurs by cell division.

BURNETT: We easily demonstrate mitosis in whole hydra by
staining them in methylene blue at pH 7 after first digesting them
with ribonuclease (1 mg. ml. for 3-5 hours). The enzyme re-
moves all cytoplasmic RNA and makes the hydra more transpar-
ent. By simply scanning the surface of the whole animal, one can
see nests of interstitial cells in synchronous division.

MACKIE: I have often seen mitosis in the cell-body part of epit-
heliomuscular cells. The fiber part is not affected. It's rather inter-
esting in silver preparations because the achromatic figure is chro-
matic and the chromatic figures is achromatic.

WOOD: Couldn't one use radioautography to trace the formation
of DNA? This might give an indication of the mitotic rate or turn-
over of cells.

FULTON : If you can figure out how to get labeled thymidine into
the animals, I'll be happy to do it. I've tried and seen nothing.

Patterns of Budding in the
Freshwater Hydroid Craspedacusta

Charles F. Lytle-

Dcpartment of Zoolofiy, Indiana University, Bloominp.t()n, Indiana

Craspedacusta sowerbii Lankester is a freshwater hydrozoan
observed sporadically in many lakes, ponds, quarries, and im-
poundments of North America. It is best known for its conspicuous
medusa stage (Fig. 1), although the life cycle also includes a
nearly transparent polyp stage, which is microscopic and devoid of
tentacles. These polyps are attached permanently to various sub-
merged objects and grow as single hydranths or more commonly
as small colonies of two to seven simple hydranths joined at their
base (Fig. 2). There is no investing perisarc on the hydranths of
C. sowerbii though a loose case of detritus can usually be seen
around the basal portion of the hydranths and the base of the
colony. This detritus is held by a mucous secretion of the epidermal
cells.

An individual hydranth is typically flask-shaped and measures
approximately 0.3-0.5 mm. in length, while a colony composed of
several hydranths may reach an overall diameter of two to three
millimeters. The hydranths may be divided roughly into four
regions: 1) a distal capitulum bearing several dozen nematocysts;
2) a constricted neck region; 3) expanded budding region; and

^This paper is contribution No. 706 from tlie Department of Zoology, Indiana Uni-
versity and is based on a portion of a thesis submitted to the faculty of Indiana
University for the Ph.D. degree. This investigation was supported in part by a pre-
doctoral fellowship CF-8674 from the National Cancer Institute, United States
Public Health Service.

2 Present address: Department of Zoology, Tulane University, New Orleans 18,
Louisiana. The author wishes to express his appreciation for the guidance and
support of Drs. Sears Crowell and Robert Briggs.

317

318

THE BIOLOGY OF HYDRA : 1961

Fig. 1. High-speed photograph of a swimming medusa. Magnification
approximately 3X.

4) a basal region by which it attaches to the substrate and/or
to neighboring hydranths.

These hydranths carry on asexual reproduction by producing
three types of buds ( Fig. 3 ) : 1 ) hydranth buds which remain
attached to the parent to form small colonies; 2) frustule or planu-
loid buds which separate from the parent and creep a short dis-
tance before developing into new polyps; and 3) medusoid buds
which are released as free-swimming medusae. Under optimal
conditions all three types of buds are formed laterally as outgrowths
of the body wall near the middle or budding region of the hy-
dranth {vide ref. 21). Differential growth in the case of hydranth
buds results in the subsequent basal attachment of adjacent hy-
dranths.

Several previous workers have observed the budding processes
of Craspedacusta polyps (3, 6-10, 12, 14-22), but only Reisinger
(20, 21) and McClary (14) have studied specific factors which
influence the production of buds under laboratory conditions.
Reisinger (20, 21) found that a sudden elevation of tempera-
ture from 20" to 25-27° could initiate medusa budding. Mc-
Clary (14) studied the growth and reproduction of polyps at

CHARLES F. LYTLE

319

Fig. 2. Macrophotograph of a polyp colony with three hydranths. The
neck and capitulum of the lower hydranth are reflexed. Magnification ap-
proximately 40X.

HYDRANTH

FRUSTULE

MEDUSA

Fig. 3. Diagram illustrating the three types of buds produced by Cras-
pedacusta polyps.

320 THE BIOLOGY OF HYDRA : 1961

four different temperatures and demonstrated that a temperature
shift was not necessary for the initiation of medusa budding. He also
observed that the three budding processes exhibited different tem-
perature optima. In his experiments, frustule production was maxi-
mal at 25°, hydranth budding was maximal at 12° and 20°, and
medusa buds were produced only at 28°.

The work discussed in the present report is concerned with the
sequence of budding in developing colonies, some effects of temper-
ature and nutrition on the growth and reproduction of polyp col-
onies, and certain physiological interactions between the different
budding processes.

METHODS AND MATERIALS

Polyps of C. sowerbii were collected on glass microscope slides
submerged in a limestone quarry pool near Bloomington, Indiana,
where populations of the medusae were known to occur regularly
(13). Laboratory stocks were established; and for these experi-
ments frustules were removed from stock cultures, isolated in Syra-
cuse watch glasses, and incubated in an 18.5° (± 1.5°) constant
temperature room. Approximately two days later the culture dishes
were transferred to shallow glass trays through which charcoal-
filtered tap water was continuously passed. In most experiments
the shallow glass trays were partially immersed in constant-temper-
ature baths. Culture water was provided from a charcoal filtration
system manufactured by the Illinois Water Treatment Company,
Rockford, Illinois (Model No. CC-24).

Polyps were fed counted numbers of oligochaete worms {Aeo-
losoma hemprichi Ehrenberg) by hand on alternate days or at spe-
cified intervals. The worms were cultured on rice-agar plates con-
taining a mixture of protozoa and l^acteria as described by Brand-
wein (2).

PATTERNS OF BUDDING

The basic pattern of development and reproduction of a polyp
colony is illustrated in Figure 4. Fifteen frustules were isolated at the

CHARLES F. LYTLE 321

211 2 C

.7 5

.50

.25

Hydranths

O o o-

Frustules

- o o-

-O O O o o o

-o o^,^^

°-, Medusae

6 8 10 12 14 16

WEEKS

Fig. 4. Budding pattern of 15 polyps reared at 27°(d=2 ). Values on the
abscissa represent the number of buds of each type produced per colony.

start of this experiment and cultured at 27° (± 2°). All frustules
had differentiated into polyps and produced an average of two
hydranths each by the end of the first week. Hydranth budding
declined during subsequent weeks but increased to a second peak
during the 13th week. Frustule production began during the sixth
week and declined to a minimum during the tenth week before ris-
ing to a new high during the 15th week. Medusa buds appeared
during the seventh week ( immediately following the decline of frus-
tule production) and were produced continuously through the 16th
week.

The basic sequence of events exhibited by these colonies was an
initial phase of rapid hydranth production, a phase of rapid frus-
tule production, and a phase of medusa budding. Secondary in-
creases in hydranth budding and frustule budding were also ob-
served during the latter portion of the phase of medusa budding.

322

THE BIOLOGY OF HYDRA : 1961

A similar sequence of events was also observed in colonies reared
at 20° and at 19-23 : At 20° {± 1°) (Fig. 5) the frequency of all
three types of budding was reduced though the same basic bud-
ding pattern was observed: an obvious initial peak of hydranth
production, a phase of rapid frustule production followed by a slight
decline, and a phase of medusa budding. At 19-23' (Fig. 6) all
three types of budding were increased and the three phases of
asexual reproduction were again clearly demonstrated.

Colonies grown at different temperatures clearly demonstrate
that under the relatively constant laboratory culture conditions three
expressions of morphogenesis occur in a sequence of distinct
phases. These activities are not mutually exclusive, but seem to ex-
hibit a clear separation between the different phases. The com-
mon basic pattern was observed at all temperatures, though certain
specific variations were noted in the duration of each phase as well

201 IC

1.0-
0.5

4.5

3.0

1.5

0.4

0.2
0

_i 1-

-O O'

Hydranths

Fr ustules

Medusae

3 4

10 II 12

Fig. 5. Budding pattern of ten polyps reared at 20 (±1). Values on the
abscissa represent the number of buds of each type produced per colony.

CHARLES F. LYTLE 323

9-23 C

H y d r a n t h s

O O- o-

Fr u St ul es

o o o o-

Medusae

10 12 14 16 18

WEEKS

Fig. 6. Budding pattern of 13 polyps reared on a water table with the
temperature rising slowly from an initial 19 to a maximum of 23 and re-
turning to 19 at the end of 16 weeks. Values on the abscissa represent the
number of buds of each type produced per colony.

as in the absolute and relative numbers of buds of each type pro-
duced at the various temperatures.

My temperature experiments also indicate the existence of cer-
tain interactions between the three budding processes. Figure 7 il-
lustrates the relationship between hydranth budding and medusa
budding. At all three temperatures there was an initial rapid rise in
the number of hydranths per colony, medusa buds appearing only
after the production of hydranths ceased or greatly declined. Me-
dusa buds were produced earliest at 20' when the total number of
hydranths produced was the smallest. Medusa buds were produced
latest in the 19-23 colonies when the total number of hydranths
was the greatest (Table 1). When subjected to statistical analysis
( analysis of variance ) , differences in the time of appearance of the

324 THE BIOLOGY OF HYDRA : 1961

o o

^^°

•

/ I9-23°C

o-
o- °

c--"— — e

6
4

«/

27t2°C

- /^•^•"

— •

2 01I°C

..^

8 10 12 14

WEEKS

Fig. 7. Relationship of colonial growth and the initiation of medusa bud-
ding at different temperatures. Values on the abscissa represent the cumu-
lative number of hydranths per colony. Arrows indicate the appearance of
the first medusa buds.

first medusa buds were found to be significant at the 95% level.

The relationship between the production of frustules and the
appearance of medusa buds is illustrated in Figure 8. Colonies at
all three temperatures exhibited an early rise in the production of
frustules and a later decline. In each case frustule budding began
after a decline in the initial rapid production of hydranth buds,
and medusa buds appeared immediately following the decline in
production of frustules.

These experiments clearly suggest that hydranth budding may
limit medusa budding, since medusa buds always appeared after
hydranth budding had declined or stopped. Furthermore, the short-
ened phase of hydranth budding at 20° is associated with the
earliest formation of medusa buds, while the extended period of

CHARLES F. LYTLE 325

TABLE 1

Age of polyp coionies at the appearance of the first medusa bud at various

temperatures.

20° (

±1°)

27° (±2°)

19-23°

45 d

ays

45 davs

72 days

54
55
56
58
62
65
57
57
60
62

75
80
78
67
68
74

Mean:

48.6

days

56.3 days

73.1 days

S.D.

2.7

2.8

1.6

hydranth budding at 19-23'^ is associated with a significant delay in
the appearance of medusa buds. It also appears that medusa bud-
ding may in turn limit frustule production since in all cases the
appearance of medusa buds is preceded by a decline in the produc-
tion of frustules.

Further evidence for this interaction between medusa budding
and frustule budding has been provided by McClary ( 14 ) . He ob-
served no medusa budding in colonies reared at 12°, 20°, and 25°.
In each of these groups there was an irregular but progressive in-
crease in the rate of frustule budding for 102 days. His 28 colonies
exhibited a rise and subsequent decline in the production of
frustules, with the decline corresponding to a maximum in medusa
budding.

To study further interactions between hydranth budding, frus-
tule budding, and medusa budding, we have investigated the effect
of increased and decreased nutrition on polyp colonies in several
ways. In the previous experiments described, frustules for the estab-
lishment of experimental colonies were taken from stock cultures

326 THE BIOLOGY OF HYDRA : 1961

■5 12

I 9 - 2 3 C

Fig. 8. Relationship of frustule budding and medusa budding at different
temperatures. Values on the abscissa represent the number of frustules
produced per colony per week. Arrows indicate the appearance of the first
medusa buds.

maintained at 23" or below. Frustules from such cultures were gen-
erally opaque as a result of large reserves of food material contained
in the gastrodermal cells ( 16, 17 ) . These food reserves occurred
in distinct cytoplasmic granules or droplets and appear to be simi-
lar to the "protein reserve droplets ' or "spherules de reserves" con-
tained in the gastrodermis of Hydra oligactis (4), Hydra attenuata
(23), and in the polyp stage of the African freshwater medusa
Limnocnida (1). Histochernical tests have indicated that these
granules or "reserve bodies" may contain RNA, DNA, protein, car-
bohydrate, and fats in varying proportions.

Frustules produced by Craspedacusta colonies cultured at
temperatures higher than 23° are appreciably less opaque, indi-
cating smaller amounts of reserve food materials. Colonies reared
from 27° frustules demonstrate a strikingly different developmental
pattern from those rean^d from frustules produced at low
temperatures.

CHARLES F. LYTLE 327

Figure 9 illustrates the de\'elopment and budding of two groups
of animals reared from 27 frustules at two different feeding rates.
The animals represented by the open circles were fed on alternate
days as in the previous experiments. They exhibited an initial phase
of rapid hydranth production followed by the initiation and rap-
id increase in frustule production— but no phase of medusa budding
and no decline in the rate of frustule production. Therefore, in
the absence of medusa budding the available food material went
preferentially into the production of frustules. The second peak of
hydranth budding during the 13th week does not appear related to
the absence of medusa budding in these animals, since a similar
secondary peak is observed at the same time in parallel groups
of animals grown at this temperature which do produce medusa
buds.

26t2°C

i=8=

H y d r a n t hs

„^:c^^° \.,^_.«=e^^

0.5

Frustules o

„ / ^~^o o o o

Medusae

8 ID 12 14

WE EKS

Fig. 9. Budding pattern of colonies reared from 27 frustules at two
different feeding rates. Colonies represented by the open circles were fed
on alternate days and those represented by the filled circles were fed every
third day.

328

THE BIOLOGY OF HYDRA : 1961

The animals represented by the filled chcles were fed every
third day. These animals demonstrate that the rate of hydranth bud-
ding is not significantly decreased by the lowered nutritional level
but that frustule production is differentially affected. Therefore,
this experiment provides direct evidence of a physiological interac-
tion between medusa budding and frustule budding and further
indicates that this interaction is at least partially nutritional.

Another experiment with different nutritional levels further illus-
trates the interactions between these three morphogenetic processes.
Colonies were reared at 23° from frustules taken from low tempera-
ture stocks until several hydranths had been formed. These colonies
were starved for approximately four weeks to deplete their nutri-
tional reserves and were divided into three groups fed at different
rates. As indicated in Figure 10, the production of hydranths showed

n/2 Daily

I Worm/ 2 Days

4 5

WEEKS

Fig. 10. Colonial growth at three different feeding rates (n = the number
of hydranths per colony at the time of feeding). Cultures maintained at
23° (±1°).

CHARLES F. LYTLE

329

30 -

I 5

^ Daily

Fig. 11. Production of frustules at three different feeding rates (n = the
number of hydranths per colony at the time of feeding). Cultures maintained

at 23 (±:1 ).

a direct and proportional increase with increased rates of feeding.
The production of frustules, however, was affected differentially
(Fig. 11). The animals at the two lower feeding rates produced
only a few frustules while the animals at the highest rate showed
a large increase in the number of frustules produced. Much of the
additional food went preferentially into the production of frustules.
The effect of these different feeding rates is summarized in Fig-
ure 12. At the lowest feeding rate there were few buds of each
type produced. At the intermediate feeding rate there was a 240%
increase in the production of hydranths over those produced at the
lowest rate of feeding and a 60^ increase in the production of me-
dusa buds. Only a 5.1% increase was observed in the production
of frustules. At the highest rate of feeding there was a further in-
crease (211.8%) in the production of hydranth buds over the inter-

330

THE BIOLOGY OF HYDRA : 1961

■

2 4 0%

Interr

nediate
60%

■

5.1 %

:^.jr--r--^^

4 7l.7y«

1 1 1

High

■

2 11.8%

■

1 2.5%

—

Hydranths

Frustules

Medusae

Fig. 12. Differential utilization of food materials by the three different
budding processes at three different feeding rates. The number of buds
at each rate is expressed as a percentage of those produced at the next
lower rate.

mediate rate of feeding, but a much smaller increase in the produc-
tion of medusa buds (12.5%). Frustule budding was tremendously
increased (471.7%). Thus at the different nutritional levels food
material was utilized differentially by the three different budding
processes as observed also in the previous experiment.

Hydranth budding appears to limit medusa budding since me-

CHARLES F. LYTLE S31

dusa buds always appear after growth has decHned or stopped,
and because the abbreviated phases of growth at 20° and 27" are
associated with the early formation of medusa buds. The longer
growth phase at 19-23 is associated with a delay in the forma-
tion of medusa buds. Also hydranth budding is the least affected of
the three types of budding by lowered nutritional level. A similar
inverse relationship between growth and medusa budding was
found in Hydr actinia by Hauenschild and in Obelia by Grell (11).
Crowell (5) also found a definite order of priority in the utiliza-
tion of nutritive substances among the several growth zones of
Campanularia when overall growth was experimentally limited.
Significantly, he observed that the formation of gonangia ap-
peared to require a high nutritive level.

Medusa budding appears to limit frustule production since in
all cases the appearance of medusa buds is preceded by a decline
in frustule budding. The production of frustules always reached an
initial peak after the completion of the initial growth phase and de-
clined prior to the appearance of medusa buds. This decline in
frustule production was most marked in the 19-23 colonies which
produced the greatest number of medusa buds, and least pronounced
in the 20" colonies where the fewest medusae were produced. In
cultures of high temperature frustules which produced no medusa
buds, there was no subsequent decline in the rate of frustule pro-
duction after the initial maximum was reached. The relationship
between hydranth budding and frustule budding was less clearly
demonstrated, but there were some indications of a similar inter-
action between these two processes also.

These experiments clearly demonstrate that temperature between
20° and 27° is not a limiting factor in the production of medusa
buds by isolated colonies in culture if sufficient food is provided,
contrary to the observations of Reisinger (20, 21) and McClary
(14). Studies on nutrition have shown that lowering of the feeding
rate within this temperature may diminish and/ or greatly delay the
production of medusa buds.

Experiments on the effect of various nutritional levels on the
budding processes of isolated colonies demonstrate that the three
budding processes are affected differentially by increased feeding
rates. At very low feeding rates, medusa budding may be reduced

332 THE BIOLOGY OF HYDRA : 1961

or eliminated, few hydranths are formed, and few friistules are pro-
duced. At intermediate rates a large proportion of the food materials
are utilized in hydranth budding and in medusa budding. Frustule
production is still low. At high feeding rates the largest portion of
the food materials is utilized in the formation of frustules and pro-
portionally less goes into the production of new hydranths and
medusa buds. Therefore these experiments provide some physio-
logical basis for the observed interactions between these three bud-
ding processes and suggest that these three morphogenetic proc-
esses are, at least in a sense, antagonistic, involving alternate
pathways for the utilization of metabolic su1:>strates. My present
hypothesis is that hydranth budding, frustule budding, and medusa
budding represent alternate morphogenetic pathways, and that the
control of budding in this system may depend upon physiological
competition for specific metabolic substrates.

REFERENCES

1. Bouillon, J. 1958. Etude monographique du genre Liiiinocnida ( Limnome-

dusae). Ami. Soc. Roij. Zool. Belg. for 1956-1957. 87: 254-500.

2. Brandwein, p. 1937. The culture of some miscellaneous small invertebrates. In

Culture Methods for Invertebrate Animals. Ed. P. S. Galtsoff et al. Com-
stock Publishing Company, Ithaca, pp. 143-144.

3. Browne, E. T. 1906. On the freshwater medusa liberated by Microhijdra ryderi

Potts, and a comparison with Linuiocodiuni. Quart. J. Microscop. Sci. 50
( N.S. ) : 635-645.

4. Burnett, A. L. 1959. Histophysiology of growth in hydra. /. Exp. Zool. 140:

281-342.

5. Crowell, S. 1957. Differential responses of growth zones to nutritive level, age,

and temperature in the colonial hydroid Campamdaria. }. Exp. Zool. 134:
63-90.

6. Dejdar, E. 1934. Die Siisswassenneduse Craspedacusta sowerbii Lankester in

monographischer Darstellung. Z. Morph. Okol. Here 28: 595-691.

7. Dunham, D. W. 1941. Studies on tlie ecology and physiology of the freshwater

jellyfish, Craspedacusta sowerbii. Ph.D. Thesis, Ohio State University,
Columbus.

8. Fowler, G. H. 1890. Notes on the hydroid phase of Lintnocodium sowerbyi.

Quart. J. Microscop. Sci. 80: 507-513.

9. GoETTE, A. 1909. Microhydra ryderi in Deutschland. Zool. Anz. 34: 89-90.

10. GoETTE, A. 1920. Uber die ungeschlechtliche Fortpflanzung von Microhydra
ryderi. Zool. Anz. 51: 71-77.

CHARLES F. LYILE 333

11. Hauenschild, C. 1954. Genetische und entwicklungsphysiologische Untersuch-

ungen an Kulturen von Hijdractinia echinata Flemm. zur Frage der Se.xu-
alitiit und Stockdifferenziemng. Zool. Jahrb., Aht. allg. Zoo/. Physiol. 64:
1-13.

12. KuHL, G. 1947. Zeitiafferfilm-untersuchungen iiber den Polypen von Craspedu-

custa sowerhii ( Ungeschlechtliche Fortpflanzung, Okologie, und Regen-
eration). Ahhandl. Senckenbeigischen NatuiiorscJwnden Ges. 473: 1-72.

13. Lytle, C. F. 1959. The records of freshwater medusae in Indiana. Pwc. Indiana

Acad. Sci. 67: 304-308.

14. McClary, a. 1959. The effect of temperature on growth and reproduction in

Craspedacusta sowcrJni. Ecology 40: 158-162.

15. MosER, J. 1930. Micwhydra E. Potts. Sitsber. Gcs. natuii. Freiindc, BerHn.

pp. 283-303.

16. Payne, F. 1924. A study of the freshwater medusa, Craspedacusta ryderi.

J. Morph. 38: 387-430.

17. Persch, H. 1933. Untersuchungen iiber Microhydra gcrnianica Roch. Z. wiss.

Zool. 144: 163-210.

18. Potts, E. 1897. A North American freshwater jelly-fish. Amer. Nat. 31: 1032-

10.35.

19. Potts, E. 1906. On the medusa of Microhydra ryderi and on the forms of medii-

sae inhabiting fresh water. Quart. J. Microscop. Sci. 50( N.S.): 623-633.

20. Reisinger, E. 1934. Die Siisswassermeduse Craspedacusta sowerbii Lankester

und ihr Vorkommen in Flussgebiet von Rhein und Maas. Nafiir am Nie-
derrhein 10: 33-43.

21. Reisinger, E. 1957. Zur Entwicklungsgeschichte und Entwicklungsmechanik von

Craspedacusia ( Hydrozoa, Limnotrachylina ) . Z. Morph. Okol. Tiere 45:
656-698.

22. Ryder, T. A. 1885. The development and structure of Microlujdra ryderi. Amer.

Nat. 29: 1232-12.36.

23. Semal-van Gansen, P. 1955. L'histophysiologie de rendodernie dc I'hydra d'eau

douce. Ann. Sac. Roy. Zool. Belg. for 1954. 85: 217-278.

DISCUSSION

FULTON: I noticed that the patterns were the same, but the
absohite numbers were very different when you grew them at 20'
versus 19 to 23 '. Was one of these in the hght and the otlier in the
dark, or anything like that?

LYTLE: No. The animals in these experiments were all grown in
an aquarium room with several large windows. No attempt was
made to alter the normal photoperiod of light and darkness.

FULTON: So far as you know the 19" to 23^^ versus the 20°
are under otherwise identical conditions, but just the temperature
varied?

334 THE BIOLOGY OF HYDRA : 1961

LYTLE: No. Unfortunately the conditions in these two experi-
ments were not precisely the same, but I do think we can say that
temperature is the most important variable here. The 20° cultures
were maintained in running water in a constant temperature bath
controlled ±1°. The 19-23° cultures, however, were maintained in
running water on a water table at the temperature of the incoming
water. During the course of this experiment the temperature rose
gradually from an initial 19 to 23 and slowly returned to 19° at
the end of 16 weeks. There was also a small diurnal variation in the
temperature, in the order of about 1 ". Furthermore, because of a
technical difficulty there was some difference in the rate of flow be-
tween the 20° experiment and the 19-23° experiment, but I doubt
that this had any great influence on our results. I believe that the
gradual rise and decline of temperature was probably more impor-
tant than the small difference in rate of flow or the actual difference
in mean temperature between the two experiments, but this has
to be investigated further.

FULTON: I see that your absolute numbers were a lot bigger
there.

LYTLE: Definitely. The large colony with 22 hydranths which I
showed at the beginning of my talk was grown on the water table
with the rise and fall of temperature (19 -23° -19"). I have never
gotten colonies this large in cultures grown under more closely con-
trolled temperatures within this range.

HAND: If I understood your summary, it sounded to me as if you
were saying something backwards. You showed that when hy-
dranth production falls off, frustule production comes on; and when
frustule production falls off, medusa production comes on. It sound-
ed as if you were saying that there was a backward action, that the
second phenomenon was somehow affecting the first one. What
were you thinking about?

LYTLE: As I stated in my talk, there appears to be a definite
hierarchy among the three budding processes. Hydranth budding
has first priority, and it is only after hydranth production slows
down that frustule production begins. Medusa budding does not be-
gin for some time after hydranth budding has ceased or greatly

CHARLES F. LYTLE 335

slowed down. In the interim there is a maximinn in tlie production
of frustules.

It appears that whenever metaboHc reserves are not being
siphoned off by hydranth or medusa budding, they become avail-
able for the production of frustules. Possibly the reason for the de-
cline in frustule production two or three weeks prior to the appear-
ance of medusa buds is that some of the reserve materials are al-
ready going into the pathway leading to the production of medusa
buds before the actual morphological appearance of buds. In
other words, the biochemical machinery is being set in motion. Sim-
ilar phenomena have been demonstrated in several other develop-
mental systems where biochemical differentiation precedes morpho-
logical differentiation.

HAND: That's fine. But as I visualized what you were thinking
about, it seemed to me that you were saying that there was a feed-
back, and there can t have been in time; I think time doesn't run
backwards.

LYTLE: Not very well, but there is another experiment we have
done which further illustrates this point. A group of animals was
reared from frustules in the normal way to obtain colonies; then
tlie feeding rate was suddenly doubled. In this case there was no
significant effect on hydranth and medusa budding, but the produc-
tion of frustules doubled. When the feeding rate was again dou-
bled suddenly, frustule production once more doul:)led, while hy-
dranth budding and medusa budding remained unaffected. There-
fore, the additional food went only into the production of frustules.

LOOMIS: We have been growing Cijanca artica in known solu-
tion for about eight months and have observed a very similar situ-
ation to the one you have described in Craspcdacusta. Thus, we
find that they will bud indefinitely if fed every day with brine
shrimp and then placed in clean water. They give no hint of form-
ing medusae under these conditions. I left one culture in the ice box
for a month, however, and then found that it had strobilized and
was now giving off medusae. The thing that is pertinent to Dr.
Hand's question, I believe, is that the new routine of starvation
and stagnation without water change stops l^udding and induces

336 THE BIOLOGY OF HYDRA : 1961

medusa formation, probably by a feedback action by inducing
partial anaerobiosis in the culture water. This problem is related to
the sexual differentiation of H. littoralis which also appears on stag-
nation, for in both animals the partial anaerobiosis of stagnation in-
duces a second pattern of differentiation to be expressed, much as
the butterfly pattern in the caterpillar becomes expressed during
metamorphosis.

LYTLE: We have done a similar experiment with the scyphisto-
mae of Amelia, although our experiments took a lot longer than
yours. We placed scyphistomae in a 5" cold room and left them
there for about six months with only an occasional feeding. Shortly
after we brought them back up into the laboratory (at 18.5 ),
they strobilized. This was the only time we have obtained strobilae
in the laboratory, although admittedly we haven't tried too serious-
ly. We did try different rates of feeding without any success, but
when we left them in the cold room they got dirty and eventually
strobilized.

CROWELL: Something similar happened with specimens of
Aurelia which we gave to students at Bellarmine College. They
tried, without success, to induce strobilization. Then, by accident,
one of the students who had quit working but had a few polyps
stored in a refrigerator, got medusae. So we have three explanations.
Starvation is important, cold is important, and neglect is important.

LOOM IS: Calculated neglect.

CROWELL: Not even calculated neglect.

Feedback Factors Affecting

Sexual Differentiation

in Hydra littoralis

W. F. LooMis

The Looniis Laboratory, Greenwich, Connecticut

We have been trying to induce sexual differentiation in Hydra
for some years now, because this instance of celhilar differentiation
is controlled externally by the water in which these little animals
live. This circumstance allows the investigator to analyze samples
from cultures that have turned sexual, and then try his hand at
recreating such water artificially. In this way, an approach to
understanding the biochemical variables that control cellular differ-
entiation becomes experimentally possible.

We have found Hydra to be nearly ideal for such a study. Thus,
any desired level of population density within a culture may be
maintained indefinitely by simply removing all the baby Hydra
that are produced daily by budding, baby Hydra being distin-
guished from their parents by the fact that they do not yet possess
buds of their own. Secondly, Hydra may be kept in simple saline
99% of the time, for they can feed on enough brine shrimp in fifteen
minutes to supply their nutritional needs for the ensuing twenty-four
hours. All the tedious routines of sterile tissue culture, therefore,
become unnecessary when this instance of cellular differentiation
is selected for study. Thirdly, the end result of cellular differentia-
tion in this system is unusually clear-cut, for even an inexperienced
observer can identify functional testes (or ova) on a Hydra if
a dissecting microscope is available. Finally, since the differentia-
tion of interstitial cells into gonadal tissue is an accessory path-

337

338 THE BIOLOGY OF HYDRA : 1961

way over and above their usual differentiation into nematocysts,
the phenomenon is reversible and sexual Hydra may be obtained
from asexual and vice versa. These various factors combined have
made the following study experimentally feasible.

Since several years' work will be reviewed in the next half hour,
permit me to use an analogy to illustrate some otherwise confusing
relationships. The analogy concerns a man who wears a little woolly
sweater. Inside his skin we know the temperature to be 98.6 F.
while the temperature of the room is perhaps 50 °F. Now the
question is: What is the temperature to which his skin is exposed?
Clearly the sweater markedly affects the answer, so that the air
in contact with his skin is nearer 98.6 the thicker, and more
impermeable the sweater. How does this relate to Hydra?

Figure 1 is a photograph of some Hydra in a Petri dish in which
a pH sensitive dye (brom cresol purple) is present as well as 0.5%
agar. This is a small amount of agar, enough to increase the vis-
cosity of the culture solution^ without making it actually gel. Ob-
serve that each Hydra is surrounded by a halo of its own making,
an area of increased acidity due to the increased pCOo adjacent
to its body surface. Each Hydra, in other words, is inside a little
woolly sweater, where the partial pressure or pCO:.. of carbon diox-
ide is neither as high as it is in his tissues proper, nor as low as it
is in the general macroenvironment of the Petri dish. This "halo
zone" corresponds then to the area inside the man's sweater. It is
the zone of partial anaerobiosis where the pCOo and pNHs are in-
creased and the pO^ and pH are decreased in a microenvironment
that is chemically quite different from that of the macroenvironment
of the Petri dish proper.

Note that the halo zone around each individual Hydra varies
with the size of the Hydra, so that larger and older Hydra are
exposed to greater degrees of anaerobiosis than smaller and younger
ones. In addition, group effects are present around Hydra that
happen to lie close together so that their halo zones overlap and
mutually reinforce each other. This group effect is clearly visible

iBVC solution composed of 100 mg./l. NaHCOa, 50 mg./l. disodiimi ediylenedia-
mine tetraacetate ('"Versene") and 100 mg./l. CaCL., dissolved in deionized water
from a Barnstead Bantam Demineralizer equipped with a red-cap Mixed Resin
cartridge.

W. F. LOOMIS

339

in Figure 1. It corresponds in our temperature analogy to the
warmth generated by a group of baby birds that huddle together
in the nest so that they create a microenvironment far warmer than
the surrounding air.

Figure 2 represents Rachevsky's formulation of such a halo
zone ( 25 ) . He postulated that if a spherical cell of radius r should
give off any metabolite such as CO^ at a rate q, then the concentra-

Fig. 1. Halo zones of partial anaerobiosis around single Hydra. These
vary in size with the size of the Hydra as well as with the closeness of ad-
jacent Hydra. See text for details.

340

THE BIOLOGY OF HYDRA : 1961

tioii of this metabolite at the center of the cell would be the sum
of four factors.

At the bottom would be the macroenvironmental background,
which in the case of pCO^ is 0.03^i atmosphere (0.22 mm. Hg) in
all samples of aerated water but 5.3% atm. in mammalian blood.

H,pNH3, pC02,etc.

Fig. 2. Rachevsky's graph of the four zones that together determine
the final degree of anaerobiosis to which the DNA in the nucleus of a
cell will be exposed. This same analysis holds for a multicellular mass of
cells such as a slime mold pseudoplasmodium. Hydra, or developing frog egg.
See Rachevsky (25) for mathematical equation that determines the profile
of this graph

Both of these backgrounds remain constant because the percentage
COo in the air (0.03%) is extremely constant while the pCO^ of the
blood is homeostatically regulated by the medullary center of the
brain.

Above the background zone in Rachevsky's graph is seen the
halo zone referred to above. This is the external gradient that forms
around any respiring cell under stagnant conditions. It reflects
both stagnation and crowding for the group effects mentioned above
also increase as population density increases.

W. F. LOOMIS 341

The third addition represents the cell membrane barrier, an
addition that is very small in the case of COo and NH3 as the lipid
cell membrane is known to be highly permeable to both these dis-
solved gases, (it is almost impermeable to the HCO.,^ and NH4+
ions that are fat insoluble ) ( 10,27 ) . For present purposes, this
third or membrane effect may be neglected.

The fourth and final addition represents the intracellular pC02
gradient that varies both with q, the respiratory rate, and r the
radius of the cell. Since cell division mechanisms keep r reason-
ably constant, we can experimentally control this fourth factor by
controlling q with a thermostat, for it has been shown that the
respiratory rate of Hydra varies logarithmically with the tempera-
ture, as well as also varying somewhat with the level of nutri-
tion (11).

The main factors that control the pCO^ in the center of a cell
according to Rachevsky are then: 1) the external macroenviron-
mental background; 2) the "halo zone" microenvironment; 3) the
barrier effect that is small if only a cell membrane is involved but
can be very large if it involves an impermeable chitinous perisarc;
and 4 ) the internal gradient.

This then was the thinking behind the various experiments re-
ported below, experiments in which we studied the effects of tem-
perature, rate of feeding, population density, stagnation, degree
of aeration etc. on the sexual maturation of Hydra. It was our
assumption that DNA in the nucleus of the interstitial cells in the
hypostome can produce RNA and specific proteins such that gon-
adal tissues form whenever their "programming" is correct in respect
to such feedback variables as pH, pOo, pNHo and pCOo etc. When-
ever the programming is not of this variety, then these same inter-
stitial cells differentiate into nematocysts due to the intrinsic pro-
gramming that, in this case, takes place wholly within the tissues
of Hydra. Only in the case of sexual differentiation does the external
halo, group, and background effects determine the outcome of the
experiment. Only this case, therefore, can be experimentally manip-
ulated by varying the external cultural conditions.

Let us examine the results of the experiment in Table 1 from this
point of view. This experiment was originally performed in 1957
(16) but has been repeated eight times since then with entirely

342

THE BIOLOGY OF HYDRA : 1961

consistent results, an exceptional record it might be said in a field
where over a score of operational factors have been shown to affect
the results. In this experiment, ten male Hydra littoralis were
grown in 15 ml. beakers in BVC solution^ that had been aerated
with oxygen in duplicate vessels 1 and 2, while vessels 3 to 8 re-
ceived increasing amounts of BVC solution that had been equili-
brated with oxygen containing 10'/ CO. gas. In all cases the Hydra
were fed daily with an excess of brine shrimp and then rinsed and
placed in clean BVC solution half an hour later when they had fed
to repletion. In addition, each vessel was rinsed a second time about
five hours later to remove any excreted material present at that
time, the pCO^ being readjusted each time the water was changed.

TABLE 1
Control of sexual differentiation in Hydra by pCO^ (From ref. 16)

Vessel

Culture water shaken with

100 per cent O2 (ml.)

Culture water shaken with

10 percent CO2 and 90

per cent O2 ( ml. )

Initial pCOo

0.0%

0.6%

2.8%

5.6%

Day

Percentage

\ of sexual forms

100

1 See p. 338.

W. F. LOOMIS

343

All vessels were kept at 25 and all newly-detached buds were
removed daily with a medicine dropper so as to maintain a con-
stant population density within the culture. Population density,
temperature, nutrition, stagnation, depth of water, surface/volume
ratio, calcium, sodium and versene concentrations, sex and species
were thus held constant.

This experiment demonstrates that under these exact conditions
pCOo is a controlling factor in the sexual differentiation of these
animals. The unusually high degree of repeatability of this experi-
ment makes it significant, therefore, that a totally different result
occurred when this experiment was repeated on a shaking machine
that shook similar but closed vessels for a few seconds every
twenty minutes day and night (Fig. 3). Under these shaken con-
ditions, the same experiment failed to yield any sexually differ-
entiated Hydra. In retrospect, this inhibitory effect of shaking is
due to the breaking up of the halo zone by mixing the micro-
environment with the macroenvironment every twenty minutes
around the clock.

Fig. 3. Automatic shaker that is turned on for 5 seconds every twenty
minutes to destroy the halo zone by mixing the microenvironment of the
Hydra with the background macroenvironment.

344 THE BIOLOGY OF HYDRA : 1961

Since the pCO- of the macroenvironment had been artificially
increased in this negative experiment, it was concluded that high
pCO^ was not the sole factor needed to induce sexual differentiation
in Hydra littoralis ( 19 ) . The nature of the postulated second factor
is still unknown; it does not appear to be simply lowered pOo or
pH, or simply increased pNHg either, or all four factors combined,
at least in any combination yet tried. Perhaps a fifth feedback factor
exists, or even a sixth, but certainly some combination of known
circumstances should be able to be brought together in the macro-
environment such that even shaken Hydra are exposed to condi-
tions equivalent to that found within the halo zone of stagnant
Hydra.

Before proceeding further, it is perhaps instructive to mention
that a powerful group effect exists within this 1957 experiment,
i.e. no sexual forms appear if one, rather than ten, Hydra are placed
in each vessel. Here is an example of the crowding-effect referred
to above in which several halo zones overlap to mutually reinforce
one another."

In contrast, single Hydra mature sexually when 0.1% agar is
added to the BVC solution in which they are grown as in Figure 1,
the viscosity being thus raised sufficiently to stop all thermal cur-
rents and hence allow extra large halo zones to form around even
single Hydra. Perhaps it is for this very reason that Puck's sludgy-
agar method enables single cells to grow in tissue culture when
otherwise groups of fifty to one hundred cells are needed as inocula
to obtain growth ( 24 ) .

With the realization that feedback factors associated with halo
zone anaerobiosis were active in this system, it became important
to develop quantitative means of measuring them. Rapid micro-
methods were consequently devised for pOo, pNHg and pCOo, a
Beckman micro glass electrode (Beckman 290-31 or 290-80) being
already available for determining the pH of unaerated 0.5 ml.
samples of water. All four methods are carried out in hypodermic

-Heisenberg's principle that the act of observing something alters the thing observed
enters here, for high levels of pCOs were first tried on ten Hydra so as to be statistic-
ally significant. Only later did it become clear that tlie ten Hydra affected each other
in a positive group effect so as to turn sexual when a lone Hydra would not, even
though he was exposed to as high pCOo as were the ten.

\V. F. LOOMIS 345

syringes to protect the samples from equilibrating with the gases
in the atmosphere. Furthermore, the tip of the needle of a syringe
may be placed at the exact point from which it is desired to take
the sample.

Our method of determining pOo has been described in detail
elsewhere (13, 15). Basically, it consists of drawing 0.5 ml. of
leuco indigo cannine reagent into a tuberculin syringe followed
by 0.5 ml. of the water sample to be tested. After mixing, the red
color that develops is measured at 586 m/x by placing the intact
syringe within the light path of a Beckman spectrophotometer, thus
avoiding all contamination of the reagent with atmospheric oxygen.

NH:i is determined in our laboratory by mixing 0.5 ml. of water
sample with 0.5 ml. of Nessler solution that has been diluted one
to live. The resulting color in the syringe is measured at 480 uifi
by the method described above for oxygen.

pCOo is measured directly by a method that has been published
elsewhere ( 17 ) . As originally described, this method required modi-
fication of a Henderson-Haldane apparatus, but this has since been
found unnecessary, the standard apparatus (New York Laboratory
Supply Co. 44250) being found sufficiently accurate for all prac-
tical purposes. One analysis takes about three minutes. It consists
of 1 ) filling a 20 ml. syringe with 10 ml. of water sample and 10 ml.
of air; 2) shaking the half-filled syringe for thirty seconds so as to
enrich the gas phase with the CO2 dissolved in the water phase;
and 3) measuring the percentage CO2 in tlie aii* phase volumetric-
ally in the Henderson-Haldane apparatus by measuring its
percentage shrinkage after exposure to NaOH.

Using these four methods, pH, p02, and pNHg, pCOo may be
determined in any given culture in less than ten minutes. Three of
the methods require only 0.5 ml. water samples while even the
fourth (the pCOo analysis) may be scaled down to 0.5 ml. if a
Scholander burette is used in place of a Henderson-Haldane appa-
ratus.'^ Alternatively, halo zone water may be prepared in large
amounts by growing many Hydra in a closed vessel that is placed
on the shaking machine described above so that the micro and
macroenvironments are mixed every twenty minutes. Sexual dif-

^Personal communication from Dr. Leonard Muscatine.

346

THE BIOLOGY OF HYDRA : 1961

ferentiation appears in such shaken cultures when the population
density is around one Hydra per ml., all Hydra being fed and
cleaned once per day.

This is our present approach to this fascinating problem. When
completed it should be possible to place Hydra in a running stream
of chemically treated water and have them turn sexual even
though all feedback between them and their culture water has been
eliminated. Figure 4 shows our apparatus for conducting such an
experiment. It was used to show definitively that increased levels
of pC02 alone were not sufficient to induce sexual differentiation
in Hydra littoralis, the conclusion being that other feedback factors

Fig. 4. Set of six syphons that allow 1-5 Hydra (in small beakers at
lower end of syphons) to be maintained in a flowing stream of chemically
known water with all feedback effects removed. The rate of water flow is
varied by the size of the hypodermic needle used as well as by the level
of water within the large bottles. A liter of BVC culture solution is added
to each bottle daily and the air space flushed out for five minutes with
whatever COo-Oi-N . mixture one desires.

W. F. LOOMIS 347

were also necessary (19). When these other factors have been
identified, and their appropriate dosage determined, it should be
possible to add the necessary components to the reservoirs of
Figure 4 and have the constantly-washed Hydra in the syphoned-
beakers differentiate sexually because they "think" they are
crowded, i.e. their ectoderm being exposed to the same conditions
found within the halo zones of a crowded culture.

Since the present multi-factor approach has evolved gradually
over several years, it may be worthwhile to review briefly the route
by which this investigation has progressed since this provides a
framework within which to discuss various important observations.

1) po,

Looking back, even our earliest observations suggested that
sexual difl^erentiation occurred under conditions of partial anaero-
biosis (11). Thus, we found that 1) a score of Hydra turned sexual
in a stagnant aquarium tank full of living Daphnia; 2) they
reverted to the asexual state a few days after the aerator of the
aquarium was turned on; 3) the shape of the container, and its
surface/volume ratio, strongly influenced the reaction as seen in
Table 2; 4) crowding Hydra almost automatically induced them
to turn sexual in BVC while 5) stagnation constituted a reciprocal

TABLE 2

Percentage of sexual forms and oxygen tension in cultures of differing
surface/volume ratio.

Percentage

Oxygen

of sexual

Depth

tension

fomis

( mm. )

(mg./l.)

after
10 days

7.3

100

8.4

100

8.6

2.5

8.7

Each culture consisted of 25 Hydra in 25 ml. BVC solution contained in a 50 ml
beaker and three sizes of Petri dishes.

348 THE BIOLOGY OF HYDRA : 1961

variable in that stagnant-but-not-ciowded cultures would turn sexual
just as would crowded-but-not-stagnant ones. Indeed this last obser-
vation explained why cultures of Hydra placed in an ice-box for
several weeks sometimes turned sexual, a method often advocated
by earlier workers who believed that they were mimicking the nat-
ural drop in temperature found in ponds in the fall of the year when
Hydra often spontaneously turn sexual. Our observations suggested
that it was the stagnation rather than the lowered temperature that
induced sexuality, for we observed other experimental cultures turn
sexual at 20°, 25° and 30° (14).

Analysis of over thirty spontaneously sexual cultures showed
that the pO^ was uniformly reduced from the 21% atm. of fully
aerated water to about 15% atm. (70% saturation with air). When
Hydra were grown in BVC solution whose pOo had been artificially
lowered this amount (by aeration with a 15% O^— 85% N^. gas mix-
ture), no sexual differentiation took place. Closer analysis (Table 2)
revealed that reduced pO^ and sexual differentiation occurred simul-
taneously but not proportionately and it was concluded that lowered
pOo was not the sole inductive stimulus if indeed it was not merely
an unimportant accompanying factor (16).

2) pCO,

Shortly after finding that lowered oxygen tension could not
substitute for partial anaerobiosis, we began to investigate the
possibility that an increase in the partial pressure — or pCOo — of
carbon dioxide gas dissolved in the culture solution might be the
inductive stimulus. This possibility was difficult to investigate at
first because no accurate means of measuring pCO^. existed. As with
oxygen tension therefore, it was first necessaiy to develop an accu-
rate and convenient determination, and as soon as this was available
( 17 ) it was found that spontaneously sexual cultures routinely
showed an increase in pCO^ from the 0.03% atm. of fully aerated
water to around 0.60% atm. Indeed pCOo levels as high as 1.2% atm.
were found in crowded cultures exposed to 100% oxygen rather than
air, and a record level of 1.43% atm. was found to occur naturally
in the hypolinmion of a neighboring fresh water pond in April ( 23 ) .

W. F. LOOMIS 349

The next step was to expose Hydra to BVC solutions whose
PCO2 had been raised artificially. Table 1 records the result of this
1957 experiment, an experiment that has been found to be highly
repeatable as described above. Taking both the group and stag-
nation factors of this particular experiment into account, it is clear
that pCOo strongly affects the reaction. Just as clear, however, is
the fact that an increase in pCO^ is not sufficient, for Hydra main-
tained in a flowing stream of BVC (Fig. 4) whose pCO^ varied
from 0.03% to 10% failed to differentiate sexually. Similar exposure
of Hydra to conditions of both high pCO^. and low pO^ failed to
induce sexual differentiation, and it was concluded that a third
factor must be operative in the system ( 19 ) .

3) pNH,

Evidence that a third factor existed induced us to assay samples
of "crowded water" for such metabolic gases as carbon monoxide,
methane, ethylene, H^S, SOo etc. Analysis by infra-red spectography,
mass spectography and gas-liquid partition chromatography failed
to show such gases to be present, only CO^. and NH,-, being detect-
able beyond the gases found in normal air. Analysis for NH3 by
the 1 ml. syringe method showed that sexual cultures usually con-
tained about 1 mg.T. NH... and that Hydra secreted large amounts
of ammonia after being fed with brine shrimp. Since the toxic level
of NH3 varied with the pH, it was concluded that the active species
was the NH3 molecule rather than the NH4+ ion as only the fonner
could penetrate the lipid cell membrane which is largely imper-
meable to polar solutes such as NH4+ (27).

Exposure of Hydra to increased levels of pNHo was accom-
plished by adding different amounts of NH4OH to buffered culture
solutions, and it was found that this variable alone and in various
combinations with increased pC02 and decreased pO^ was unable
to induce sexuality in Hydra at least under the conditions tried to
date. One insight came from these experiments, however: it became
clear that Hydra release the salt ammonium bicarbonate into their
halo zone and that this buffer is equivalent to NaHCOg which,
as we will see below, strongly affects the system.

350 THE BIOLOGY OF HYDRA : 1961

4) pH

Generally speaking, Hydra differentiate sexually above pH 7,
the optimum being about pH 8. Since a pCO^, of 0.5%— 1% atm. is also
required, the original pH of the unused culture solution must either
be about pH 9 in weakly buffered solutions such as BVC, or else
about pH 8 when strongly buffered with sodium bicarbonate, tris
(hydroxmethyl) aminomethane, or Versene, which is a buffer as
well as a chelating agent since it is an organic amine. In addition,
we have seen that Hydra produce their own buffer — NH4HCO;i —
in sufficient amounts to be very important. For example, the water
from a dense culture of Hydra may contain as much as 5 mg./l.
NH,, (i.e. 10 mg./l. NH4OH). At pH 8, this would be almost entirely
in the form of ammonium bicarbonate, this concentration of ammo-
nia having served to neutralize COo that otherwise would have
created a pCOo of 0.80% atm. Since this newly formed ammonium
bicarbonate now serves as so much extra bicarbonate, it is clear
that the liberation of NH-; during digestion affects the pH, the
bicarbonate concentration and both the pNH:j and the pCO:-. Since
all determinations of pCO.. from pH depend on knowing the bicar-
bonate concentration ( Henderson-Hasselbalch equation), it follows
that all such measurements are suspect in crowded cultures since
these can spontaneously increase their bicarbonate concentration
through this mechanism. The direct method of measuring pCOo
described above, of course, is not subject to this error.

The powerful effect of buffer concentration is seen in the fact
that for an entire year we failed to produce any sexual Hydra when
they were grown in 70 mg. T. CaCL.; 350 mg/1. NaCl; and 10 mg./l.
NaHC03 (12). When the NaHCO., was increased tenfold to 100
mg. 1. (14), almost every culture in the laboratory turned sexual
( 21 ) . In this connection, it is interesting that Dr. Park never observed
any sexual Hydra over a period of six years while using an unbuf-
fered culture solution composed of 0.4 mg. 1. KCl; 10 mg./l. NaCl;
and 4.8 mg. T. CaCL. We have confirmed her observations and also
found that Hydra rapidly turn sexual when 100 mg./l. NaHCOa
is added to her solution.

I would now like to describe in some detail a convenient method
of growing Hydra (and other hydroids) under constant conditions

W. F. LOOMIS 351

of pH, pOo, pNH;5 and pCO^. In essence, the method consists of
setting these variables in the water of uncrowded cultures twice a
day. These twice-daily water changes are usually carried out thirty
minutes and five hours after the cultures have been given their
daily feeding of brine shrimp. In addition, the closed culture vessels
are left on a shaking machine that shakes them strongly every
twenty minutes (Fig. 3). Continuous mixing mechanisms, such as
tissue culture roller tubes have been found to be either damaging
to Hydra or not strong enough to break up the halo zone.

Figure 5 illustrates the method used in setting the pCOo of a
series of Hydra cultures. Between one and ten Hydra are placed
in a 30 ml. Pyrex weighing bottle with an interchangeable ground
glass stopper within which are placed 25 ml. of culture solution
which leaves 5 ml. of air space. Into such vessels are injected 1-10
ml. BVC culture solution whose pCO^. has been set at 10% atm. by
bubbling it for ten minutes with the gas from a Matheson tank of
compressed air containing 10% COo. This bubbling is carried out
about once a week, for the CO^-enriched BVC solution can be stored
in 100 ml. syringes as shown in Figure 5 since CO^ does not escape
from solutions stored in this fashion. Figure 5 also demonstrates
the method of filling such 100 ml. syringes from the bottom of a
500 ml. graduate in which the bubbling is carried out.

The daily routine, therefore, consists of filling a 30 ml. dispens-
ing syringe from the 100 ml. syringe-reservoir and injecting 0, 1, 2,
3, 4 ml. etc. of this solution through a long needle into the bottom
of half filled culture vessels and then bringing their total fluid
content to 25 ml. When these vessels are shaken, the gas and water
phases equilibrate and then remain constant. The actual level of
pCOo in the various cultures is determined the next morning by the
direct method described above.

The great solubility of NH-.. makes pNH;, easy to adjust for all
that is necessary is to add varying amounts of NH4OH to aliquots
of the culture solution whose pH has been set with a bufl^er system.
Culture solutions containing various concentrations of NH4OH
are thus prepared before the start of an experiment and then stored
in capped gallon jugs. Whatever buffer is desired is also included
in such solutions, provision being made for the change of pH that

352

THE BIOLOGY OF HYDRA : 1961

comes from the later injection of culture solution that has been
enriched with dissolved COo gas.

The final step in the twice daily setting of pH, pOs, pNHg and
pCO^ involves setting the oxygen tension. Since oxygen is only

*>v s^^«

Fig. 5. The pCOo of a culture is adjusted by injecting a calculated
amount of culture solution that has previously been bubbled for ten
minutes with gas from a tank of compressed air containing 10% CO.. (see
tank behind technician). This COj-rich water is stored in the large 100 ml.
syringes on the bench for later use. Such syringes are filled with a long
glass tube from the bottom of the tall cylinder as illustrated.

slightly soluble in water, pOo is set by adjusting the air phase
and then allowing the shaking machine to keep the air and water
phase in equilibrium. In practice, a 100 ml. syringe is filled with No
from a tank and then partially emptied and refilled to 100 ml. witli
room air, the syringe thus containing whatever dilution of air in
nitrogen that one desires. A needle is attached to the syringe and the

W. F. LOOMIS 353

tip of the needle slipped under the ground glass stopper of the cul-
ture vessel. Since the air space within the vessel is only 5 ml., it is
clear that emptying the 100 ml. syringe into this space flushes it with
twenty times its own volume and so sets the air phase to whatever
percentage of oxygen one desires.

Summarizing then, buffer and ammonia concentration are
arranged at the start of an experiment by preparing sufficient
amounts of appropriate culture solution to last for the duration of
the experiment. Then pCOo-enriched water is injected at each
water change from a syringe and the air space blown out with
No-diluted air. The vessels are then left on a shaking machine that
agitates them every twenty minutes.

Proof that pH, pO., pNH., and pCO. do not change is obtained
by analysis. For this, the pH and pCOo are first determined and
the results plotted on semi-log paper as in Figure 6. After this it is
an easy matter to follow the cultures with a daily pH which yields
their pCOo as taken from the logarithmic calibration curve.

The above system has been gradually evolved over several years.
With its aid, we can now analyze and control most of the seemingly-
magic variables that affect this system. How can the mere change
of one Pyrex vessel for another completely alter the results (Table
2)? A second rinse? Leaving the cultures over the weekend? Chang-
ing the bicarbonate concentration? Aerating the cultures? These
and many other operational variables are reflected in the changed
values found in our four feedback variables. Some of them are listed
in Table 3 where they are correlated with Rachevsky's four zones
as well as with their equivalents in the analogy of the man in the
little woolly sweater.

Perhaps further work will show that sexual differentiation can
be chemically induced by appropriate levels of pH, pOo, pNH^
and pCO^, i.e. that it is just a matter of finding correct dosage
levels. Alternatively, it may be that further feedback xariables are
involved that will need identification, analytic quantitation and
artificial application before Hydra will differentiate sexually in a
no-feedback system. To date we have run experiments that appar-
ently eliminate: carbon monoxide, ethylene, carbonic anhydrase,
biotin, folic acid, lactic acid, as well as the possibility that a diurnal

354

THE BIOLOGY OF HYDRA : 1961

pCOa in % Atm
on Log Scale
5.00
4.00

3.00
2.00

1.00

.50
.40

.30
.20

.10

.05

7.0

-Solution A

\, ^

?fv

Solut

on B

■\

7.2

7.4

7.6

7.8

8.0

Fig. 6. Logarithmic calibration curves relating pH and pCO., in two solu-
tions (25 ): (1) 2 10 • M NaHCO,; 5 10"^ M CaCL„"and (2) BVT
solution prepared as described in Loomis and Lenhoff (21). Values of pC02
obtained by method of Loomis (17).

cycle of alternating high and low levels of pCOo is required for
differentiation to occur.

Before concluding this presentation, I would like to broaden the
discussion by suggesting that pH, pO., pNH,. and pCO. affect
many biological systems other than Hydra. Some of these systems
have been mentioned in previous publications (14, 16, 18), but
preliminary experimental work in this laboratory suggests that the
following phenomena are controlled by one or more of these four
feedback variables :

1. Tentacle number and rate of bud growth in Hydra (20).

W. F. LOOMIS

355

2. Inhibition of Tubiilaria regeneration: Even an amputated

hydranth can inhibit regeneration in adjacent Tubularia
stems (5, 7, 28 ) . This may result from the low intracellu-
lar pH that results from high background pCOo produced
by bacterial decomposition (and possibly intrinsic respi-
ration) of the amputated hydranth.

3. The Fulton effect in Cordylophora:

Fulton observed that Cordylophora "may be grown at-
tached to microscope slides slanted in 100 ml. beakers.
Such cultures may be grown to considerable density,

TABLE 3

Rachevsky zone
( see Fig. 2 )

Analogous zone

Factors affecting sexual differ-
entiation in hydra

Internal gradient

Body temperature

Factors that affect the metabolic

rate q: temperature, nutrition,

etc. see (11)

Factors affecting the radius r:

size of individual hydra, which

varies with age, also species of

hydra

Barrier zone

(Cell membrane,

perisarc, etc. )

Permanent insulation
such as fur

Genetic differences between
strains, species, and genera of
hydra and hydroids. May vary
in thickness of perisarc, etc.

External gradient

Halo zone

Variable insulation

such as a

woolly sweater

Effect of stagnation

shaking

second water rinse

crowding (popula-
tion density)

agar (viscosity)
Fulton effect

Background zone

Room temperature

Effect of aeration

shape of vessel
surface/volmne
absolute depth
other respiring Ufe
degree of stagnation
bicarbonate concen-
tration
Versene concentra-
tion
pH, pNHs, PO2 pCO.
of culture water

356 THE BIOLOGY OF HYDRA : 1961

whereas cultures grown in the bottoms of dishes quickly
become necrotic." (6). This striking "position effect" in
CordylopJwra contrasts with Hydra which can grow
equally well on the bottoms or sides of dishes. Preliminary
results suggest that growth of Cordylophora is especially
sensitive to the self-induced acidity present in the halo
zone and that such zones are largely prevented from
forming on slide-grown cultures by thennal currents
( these can be made visible with methylene blue or other
dyes ) . Experimental elevation of the pCOa in shaken cul-
tures gradually inhibits Cordylophora growth whenever it
is sufficient to lower their pH below about 6.7, the actual
pCO:- varying with the buffering capacity of the solution
employed.

4. Strobilization in Cyanca arctica.

This organism buds indefinitely when fed and placed in
clean water daily. One culture strobilized and produced
many medusae after being left untouched for a month in
an ice-box at 12'.

5. Spiral persons in Hydractinia and Podocoryne.

Braverman has shown that spiral zooids of Podocoryne
appear on the rim of hermit crab shells only if the shell
is inhabited by a living hermit crab (4). He noted that
spiral zooids never form on colonies grown on glass slides
in the laboratory, an observation that we have confirmed
on Hydractinia. Spiral zooids appear all over slide-
grown Hydractinia cultures exposed to a pCO^ of 2%
atm.: a result that suggests that CO2 coming from the
respiration of the hermit crab is the stimulus that creates
spiral zooids on lips of hermit crab shells.

6. Parthenogenetic reproduction in Daphnia.

Both Daphnia longispina and Daphnia magna fail to re-
produce parthenogenetically in aerated water while doing
so in water whose pCO:.. is 1% atm. and whose pOo is 5%
atm. Daphnia are thus neither aerobic nor anaerobic
organisms, but like microaerophilic ("little air") bacteria,
they require partial anaerobiosis to live. This fact ex-

W. F. LOOMIS 357

plains their usual habitat which is the partially anaerobic
environment of the hypolimnton (2) as well as their
demand for the microaerophilic environment of a soil-
manure culture ( 1 ) .

Amoeboid motion.

Amoeba proteus and Chaos chaos are both far larger than
the usual metazoan cell. Their central protoplasm would
become extremely anaerobic if it did not liquify and then
flow peripherally in long pseudopods with a high surface/
volume ratio. The possibility that amoeboid motion results
from this automatic gel-to-sol transformation under the
high pCOo (and consequent low pH) existing in the
center of these animals is supported by the experimental
finding that a pCOo of 20% atm. "melts" their pseudopods
back into their bodies so that they become spherical in
form. With time, continued respiration would re-establish
the inward-outward gradient of pCO^, and hence normal
amoeboid motion should recommence. In fact, it does
just this, but only if oxygen is present. Furthermore,
amoebae can crawl for days in the presence of 10 ~' M
dinitrophenol which is known to uncouple oxidation from
phosphorylation at this concentration (22) and so sug-
gests that it is the COo from respiration rather than the
ATP that is important in amoeboid motion. Finally,
Pantin showed with neutral red that the anterior and sides
of an advancing pseudopod were bright red (acid) and
that the color was stronger the more active the pseudo-
pod. This was especially the case with eruptive pseudo-
pods.

The acrasin problem.

The slime molds grow as many separate amoebae and
then aggregate by chemotaxis in response to the mutually-
produced but unidentified chemical named "acrasin" by
Bonner (3). Some preliminary experiments suggest that
the highly labile stimulus may be simply pCOo. With this
goes the possibility that high levels of pCOo in the
center of the multicellular pseudoplasmodium stimulate

358 THE BIOLOGY OF HYDRA : 1961

cellulose deposition and hence stalk formation in all cells
that become buried in the central mass (see 26). Such
a mechanism would explain how identical slime mold
amoebae can differentiate into completely different types
of cells once they have aggregated into a single multi-
cellular mass whose central or medullary pCOo and pH
are utterly different from what they are in the peripheral
cortex.
Returning now to Rachevsky's formulation, we find that it
applies to both isolated single cells ( as he originally meant it to be )
and to multicellular masses of cells such as hydroids and slime mold
pseudoplasmodia. In fact, the great permeability of cell membranes
to both NH3 and CO^ serves to unite such multicellular masses into
one supercellular field or gradient of pCOo and pNH.j. Goldschmidt
has stated (8) that "the most difficult and most neglected of all
basic fields of morphogenesis is that of supercellular integration,"
and it is of interest therefore that Rachevsky's ideal formulation was
developed for an isolated cell but fits the facts in differentiating
metazoan tissues as well.

For example, Rachevsky's graph clearly brings out the great
morphogenetic importance of a perisarc, that chitinous non-living
envelope that surrounds the living coenosarc of some hydroids ( 29 ) .
In our analogy, a perisarc would correspond, not to a removable
woolly sweater, but to a permanent coat of fur as seen in northern
animals. Both types of insulation serve to build up the internal
temperature gradient, but a perisarc is permanent while the halo
zone "woolly sweater" is dependent on the presence of stagnant
conditions.

In this connection, Cordijlophora possess a perisarc and differen-
tiate sexually in May and June even while growing in the fully
aerated spillway of Nye's Pond near Woods Hole.^ In this case,
the perisarc probably insulates the animal sufficiently to allow par-
tially anaerobic conditions to form inside its tissues as the tempera-
ture and food supply gradually increase their metabolic rate in
late spring. In contrast to this. Hydra have no perisarc and turn
sexual in the late fall when the temperature and food supply are

■^observed to be sexual in 1960 by C. Fulton and 1961 by W. F. Loomis.

W. F. LOOMIS 359

dropping but when winter anaerobiosis in the pond is steadily
increasing (9).

Seen from this angle, a Hydra is nearly as naked as a clone of
cells growing in tissue culture. Having no perisarc, it is exposed
to its external milieu in feedback fashion just as is a pseudo-
plasmodium or a developing frog egg. In each of these cases,
morphogenetic gradients form and gradually shape the once-
identical cells into diverse populations of differentiated cells. The
purpose of this paper has been to show how these gradients may
be approached both conceptually and experimentally.

REFERENCES

1. Banta, a. M. 1959. In Culture Methods for Invertebrate Animals by F. E. Lutz,

P. L. Welch, P. S. Galtsoff and J. G. Needham. Dover Pubs., Inc., New
York, page 207.

2. BiRGE, E. A. 1903. The themiochne and its significance. Trans. Am. Micro. Soc.

25: 5-33.

3. Bonner, J. T. 1960. Development in the cellular slime molds: The role of cell

division, cell size and cell number. In Developing Cell Systems, 18th
Growth Symposium, D. Rudnick, Ed., Ronald Press, New York.

4. Braverman, M. H. 1960. Differentiation and commensalism in Podocoryne

carnea. Am. Midland Naturalist, 63: 223-225.

5. Fulton, C. 1959. Re-examination of an inhibitor of regeneration in Tuhularia.

Biol. Bull, 116: 232-238.

6. Fulton, C. 1960. Culture of a colonial hydroid under controlled conditions.

Science, 132: 473-474.

7. GoLDiN, A. 1942. A quantitative study of tlie interrelationships of oxygen and

hydrogen ion concentration in influencing Tuhularia regeneration. Biol.
Bull, 82: 340-346.

8. GoLDSCHMiDT, R. B. 1955. Theoretical Genetics. University of California Press,

Berkeley, California.

9. Hutchinson, G. E. 1957. A Treatise on Limnology. Vol. I. Wiley & Sons. New

York, page 627.

10. Jacobs, M. H. 1920. The production of intracellular acidity by neutral and

alkaline solutions containing carbon dioxide. Am. J. Physiol, 53: 457-463.

11. Lenhoff, H. M. and W. F. Loomis. 1957. Environmental factors controlling

respiration in hydra. /. Exp. Zool 134: 171-182.

12. Loomis, W. F. 1953. The cultivation of hydra imder controlled conditions.

Science, 117: 565-566.

13. Loomis, W. F. 1954. Rapid microcolorimetric determination of dissolved oxygen.

Anal Chem., 26: 402-404.

14. Loomis, W. F. 1954. Reversible induction of sexual differentiation in Hydra.

Science, 120: 145-146.

15. Loomis, W. F. 1956. Improved rapid colorimetric microdetennination of dis-

solved oxygen. Anal Chem., 28: 1347-1349.

360 THE BIOLOGY OF HYDRA : 1961

16. LooMis, W. F. 1957. Sexual differentiation in hydra: Control by carbon dioxide

tension. Science, 126: 735-739.

17. LooMis, W. F. 1958. Direct method of determining carbon dioxide tension.

Anal. Chem., SO: 1865-1868.

18. LooMis, W. F. 1959. Feedback control of growth and differentiation by carbon

dioxide tension and related metabolic variables. Chapter 9 in Cell, Organ-
ism and Milieu, 17th Growth Symposium, D. Rudnick, Ed., Ronald Press,
New York.

19. LooMis, W. F. 1959. Further studies on cellular differentiation in Hydra. Fed.

Proc, 18: 1092.

20. LooMis, W. F. 1959. The sex gas of hydra. Sc. Am. 200: 145-156.

21. LooMis, W. F., and H. M. Lenhoff. 1956. Growth and sexual differentiation of

hydra in mass culture. /. Exp. Zool., 132: 555-574.

22. LooMis, W. F., and F. Lipmann. 1948. Reversible inhibition of the coupling

between phosphorylation and oxidation. /. Biol Chem., 173: 807-808.

23. LooMis, W. F., and W. F. Loomis, Jr. 1960. Constancy of the pCO- in the

ocean. Biol. Bidl, 119: 295.

24. Puck, T. T., P. I. Marcus, and S. J. Cieciura. 1955. Clonal growth of mam-

malian cells in vitro: Growth characteristics of colonies from single HeLa
cells with and without a "feeder" layer. /. Exp. Med., 103: 273-284.

25. Rachevsky, N. 1960. Mathematical Biophysics, Physico-Mathenmtical Founda-

tions of Biology. Dover Pubis., Inc., New York. Figure 1., page 32.

26. Raper, K. B., and D. I. Fennell. 1952. Stalk formation in Dictyostclium. Bull.,

Torrey Botanical Club, 79: 25-51.

27. Robin, E. D., D. M. Travis, P. A. Bromberg, and C. E. Forkner, Jr. 1959.

Ammonia excretion by mammalian lung. Science, 129: 270-271.

28. Tardent, p. E. 1960. Principles governing the process of regeneration in

hydroids. Chapter 2, in Developing Cell System, 18th Growth Symposium,
D. Rudnick, Ed. Ronald Press, New York.

29. Waterman, T. H. 1950. In Selected Invertebrate Types. F. A. Brown, Jr., Ed.,

Wiley & Sons, New York, page 89.

DISCUSSION

MUSCATINE : Have you ever turned green hydra sexual?

LOOMIS: Only once, even though I have grown them in "sludgy
agar" and under the other conditions that make H. littoralis turn
sexual. That one time was after they had been neglected in a stag-
nant aquarium for a week or so.

KLINE: Two questions. If you carry out the pC02 experiment
in the low bicarbonate medium, what kind of results do you get?
Also, I have heard discussion of a CO^-bicarbonate equilibrium in
which the two compounds were considered to be interchangeable.
Will you comment on this?

LOOMIS: If the pCOo experiment is done in a low bicarbonate

W. F. LOOMIS 361

medium, the Hydra die for the pH becomes too acid. I do not
have the full answer yet about just how much alkalinity, increased
pCO., and the bicarbonate ion matters. Probably all three do, at
least indirectly through the Henderson-Hasselbalch equation, but
also perhaps each directly in its own right. An additional complica-
tion lies in the fact that crowded Hydra liberate ammonium bicar-
bonate in significant amounts and this salt can substitute for sodium
bicarbonate as a pH buffer.

Now you asked about the pCOs-bicarbonate equilibrium. Well,
aerated bicarbonate-Versene-calcium solution does not work, hence
the bicarbonate alone is not enough. What is needed in addition
it seems is the CO:, that comes from the respiration of crowded
Hydra.
KLINE: Will there always be an equilibrium between the two?

LOOMIS: Yes, a three-way equilibrium between pH, pCOo, and
bicarbonate. You can set each one in an experiment and hold it
constant while you vary the other two reciprocally, and you can do
this for each of the three variables in turn. In this way, you can
determine the role of each variable independently. I hope to do
this as soon as I have the "halo zone" under control. The final
answer will come when a solution can be prepared that will turn
isolated single Hydra sexual without the need for any crowding
or stagnation.

LENHOFF: I would like to propose a mechanism showing one
way in which CO^. can play a role in controlling sexual differentia-
tion. This \iew emphasizes that COo is an important metabolite
needed for synthetic processes of the cell. First we must recognize
that aside from producing CO. the Krebs cycle also serves at
least two other major functions; it provides hydrogen atoms for
energy production; and, of equal importance, it provides carbon
skeletons for the synthesis of major portions of other molecules,
such as some unessential amino acids and pyrimidines. Thus, when
unusual demands are put on the cell's synthetic machinery, such as
occurs during cellular differentiation, the keto acids may be pulled
out of the Krebs cycle to give, for example, amino acids for protein
synthesis. That is, «-ketoglutarate and oxaloacetate yield glutamate
and aspartate on amination. When under these demands, the cell

362 THE BIOLOGY OF HYDRA : imi

has to keep the Krebs cycle operating by replacing the di- or tricar-
boxyllic acids. This is usually taken care of by the Wood-Werkman,
Utter, or malic enzyme pathways, all of which require a fomi of
pyruvate and COo. In uncrowded cultures, however, hydra, as well
as bacteria and tissue culture cells, probably lose much of their
metabolic COo to the environment. It would seem that these cells
would have difficulty in resynthesizing the dicarboxyllic acids unless
the partial pressure of CO2 was increased either naturally, as in
crowding, or artificially using known gas mixtures.

If this were true, then by exposing starved H. littoralis to C^^O.,
one might expect the C^^ to be found mostly in glutamate and
aspartate. This proved to be the case (Lenhoft, H., 1959, Exptl.
Cell Research 17, 570-574). Alanine, which comes from pyruvate,
and therefore would not be expected to incorporate C^^Oo, was not
labeled. Furthermore, the C^^ was localized in cnidoblasts, which
are known to be active in the synthesis of nematocyst protein in the
starved animal. The large amount of RNA-rich endoplasmic reti-
culum as shown in Slautterback's electron micrographs of cnido-
blasts, is another indication of protein synthesis. Also, when we in-
duced sexual difterentiation in starved Hydra in the presence of
C^^Oj, much of the C^^ was concentrated in the testes and the
ovaries of sexual Hydra.

Thus, the partial pressure of CO- may take on special impor-
tance in animals such as hydra that readily lose COo to the en-
vironment. The increased pCOo would serve to drive the reactions
forming the dicarboxyllic acids, thereby maintaining the continued
operation of the Krebs cycle and thus regulating the activities of
the cell.

LOOMIS: Yes, your radioautographs are very dramatic, showing
how the C^^ is concentrated in the growing testes and ovaries as
well as in the cnidoblasts.

Added in proof: Two recent articles on pCOs are: Goddard, D. R. 1960. The
biological role of carbon dioxide. Anesthesiology, 21: 587-596, and Loomis, \V. F.
1961. Cell differentiation: a problem in selective gene activation through self-
produced micro-environmental differences of carbon dioxide tension, in Biological
Structure and Function, First lUB/IUBS Joint Symposium, O. Lindberg and T. W.
Goodwin, eds.. Academic Press, London (in press).

Apparent Rhythmicity

in Sexual Differentiation

of Hydra littoralis

Helen D. Park

Laboratory of Physical Biology, National Institute of Arthritis and Metabolic
Diseases, National Imtitiites of Health, Bethesda 14, Maryland.

Hydra have been studied in our laboratory for many years, first
by Dr. Harold Chalkley, then by Dr. George Daniel, and for the
past seven years by myself. Dr. Loomis' early studies (3, 4, 5) on
Hydra littoralis, which he has just reviewed for us, are especially
interesting to me because Dr. Daniel and I maintained mass cul-
tures of a clone of Hydra littoralis in our laboratory from 1950-1956
with only one three-week period in which any sexual Hydra were
observed. These cultures were maintained in a solution containing
iOO mg. NaCl, 4 mg. KCl, 48 mg. CaCL per liter of double dis-
tilled HoO, the last distillation being from glass. The cultures,
unfortunately, were discarded in 1956.

In 1958, however, we obtained a culture of H. littoralis from Dr.
Edward Kline of the Armed Forces Institute of Pathology.
These were Loomis stock and had been cultured in BVT ( 100 mg.
NaHCOs and 50 mg. Versene per liter of tap H.O ) . We established
a clone from a spermary-bearing individual and for three years
have maintained in BVT mass cultures derived from this single
male. Daily except Sundays we allow the cultures to feed on an ex-
cess of Artemia lar\'ae for 30-60 minutes, rinse them with tap H^O,
and replenish the BVT. The laboratory temperature is 24° ± 2°.

Soon after setting up the clone, we noticed that on some days
sexual forms were abundant; on other days they were difficult, if not

363

364

THE BIOLOGY OF HYDRA : 1961

impossible to find. This seemed interesting and puzzling enough
to warrant quantitative investigation. Therefore, Hydra were picked
at random from stock and placed in groups of 400 in finger bowls
10 cm. in diameter, containing 150 ml. of BVT. This gave a fluid
depth of approximately 2 cm. These cultures were maintained as
described above except that before each feeding we made total
counts and counts of the sexual forms, and after each feeding we
randomly discarded animals to keep the population at 400.

Figure 1 shows the percentages of sexual forms observed in a 400
Hydra culture over 200 days (Cf. 6). The ordinate value of each
point is the average of four days' measurements. This culture was
kept in the same bowl throughout. For the past 12 months, however,
we have been changing all cultures to clean dishes and counting
once a week. The percentages of sexual forms observed weekly have
ranged between 10 and 55. I think you can see from Figure 1 why
we began to think in terms of rhythmicity of sexual differentiation.

This type of curve is interesting, but tells us nothing about what

60 80 100 120 -140

DAYS AFTER START OF CULTURE

200

Fig. 1. Rhythmicity of sexual differentiation in a culture of Loomis stock
Hydra maintained at constant population density for 200 days. Each ordinate
represents the mean of four days observations.

HELEN D. PARK

365

an individual Hydra contributes to the curve. Howe\er, we now
have records of some 40 to 50 individual, isolated Hydra for peri-
ods of 80-240 days (Cf. 7). Hydra bearing spermaries were select-
ed from the stock cultures. Each Hydra was placed in 10 ml. of
BVT in a 30 ml. beaker and maintained as described above ex-
cept that buds were removed within 24 hours after separating from
the parents.

Figure 2 shows the alternating sexual and asexual periods of 10
individuals left in their beakers throughout the observation per-
iod. Hydra #1, #4, and #5, whose records do not run 200 days,
were discarded because they began to grow small and became trans-
parent even though they appeared in ingest food and were pro-
ducing buds. As can be seen, 6-10 days after isolation, each
Hydra lost its spermaries. In the next 4-23 days each again diflFer-
entiated sexually. This sexual period was followed by alternating

HYDRA:

60 80 100 120

DAYS AFTER ISOLATION

200

Fig. 2. Alternating sexual and asexual periods of 10 isolated, individual
Loomis stock Hydra observed for 200 days. Hydra 1, 4 and 5 discarded be-
cause of unhealthy appearance. Solid sections of bars, sexual periods;
hatched sections, asexual periods.

366 THE BIOLOGY OF HYDRA : 1961

asexual and sexual periods varying greatly in length. Since my ar-
rival here, Dr. Robert Bryden has called my attention to Ito's ( 2 ) ob-
servation of such alternating sexual periods in Hydra magnipapilata.

A striking characteristic of our isolated Hydra was the extreme
variability in duration of sexual periods for a given Hydra and
from Hydra to Hydra. For example, Hydra #6 went through 10
cycles while Hydra #2 went through only 3. Hydra #2 holds a rec-
ord in our laboratory for sustained production of spermaries— 103
days. I mention this in view of Brien's (1) statement that main-
tained sexuality results in death.

I would like, before we leave these isolated Hydra, to give you
a bit of information which is not relevant to the main theme of this
paper, but which I obtained in order to answer a recurring ques-
tion, "Does sexuality have any effect on l^udding rate?" We now
have an answer for 22 of these isolated Hydra involving a total of
69 sexual and 75 asexual periods after the initial sexual period. The
budding rate per Hydra per day while sexual was 0.64 ± .08 and
the rate while asexual was 0.78 ± .10. The difference is not statisti-
cally significant, and we conclude that sexuality does not affect
budding rate.

We have known since we established our mass cultures that they
are not sterile. We know that there are at least three kinds of pro-
tozoa, at least five kinds of bacteria; there are some molds. We have
not attempted sterile techniques. Some time ago I was quite im-
pressed by the possibility that the rhythmic nature of sexual dif-
ferentiation might be related to a rhythm in some other organism
or organisms in the cultures. We thought, therefore, that we would
see if keeping individual, isolated Hydra "cleaner" would have
any effect on the duration of sexual periods. Accordingly, we left
half of the individuals in the same beakers for 100 days and changed
the other half to clean beakers daily except Sunday. Other pro-
cedures were the same as those described above.

Figure 3 shows the results of two replicate experiments. Again,
we found great variation in the lengths of sexual periods for a given
Hydra and from Hydra to Hydra. The mean number of sexual
days per Hydra in the changed group was 44 ±12 and in the un-
changed group, 50 ± 13. The difference is not statistically significant
and indicates that the rhythm of sexual differentiation was not

HELEN D. PARK

367

intimately related to the presence of other organisms in the dishes.

Before going on to describe some of our most recent work, I

want to mention our experiences with clones of the Loomis stock

EXP I

Hydra DISHES CHANGED DAILY

^ ^^ mssi. fsm

Hydra
#7|

Expn

DISHES CHANGED DAILY

*im^///y^m^.

DISHES NOT CHANGED

#9|

I 1

DISHES NOT CHANGED

*^^t/yMy///.mm'yy//y/////////y^^^^^ #12^

0 10 20 30 40 50 60 70 80 90 100

DAYS AFTER ISOLATION

10 20 30 40 50 60 70 80 80 100

DAYS AFTER ISOLATION

Fig. 3. Comparison of sexual periodicity in individual, isolated Hydra left
in same dishes for 100 days with perodicity in comparable Hydra changed to
clean dishes daily. Solid sections of bars, sexual periods; hatched sections,
asexual periods.

transferred from BVT to Daniel-Park saline. Over the past three
years we have started, at intervals, perhaps a dozen such clones.
All have become sexual after 15-25 days. Our present clone has
now been maintained for about two years and the weekly counts
show that the percentages of sexual forms vary between 23 and 68,
percentages that are quite comparable to those for the cultm'es in
BVT. We are unable to explain the difference between the asexual
clone of Hydra littoralis in Daniel-Park saline from 1950-1956
and the clone of Loomis stock over the past three years.

About five months ago I decided that we had worked exclusively
with the Loomis stock of Hydra long enough. We, therefore,
ordered a culture of H. littoralis from the Carolina Biological Sup-
ply Company, thinking that perhaps H. littoralis from another part

368 THE BIOLOGY OF HYDRA : 1961

of the country might be different from the Loomis stock in its pat-
terns of sexual differentiation.

The Carohna Hi/dra arrived in pond water. We were immedi-
ately impressed by their large size when compared with the Loomis
stock. They were, and have continued to be, three to four times
larger. We need to ha\'e the hydra positively identified, but whether
they are H. littoralis or not, we have learned something from them.

The Hyclra were asexual when they arrived. We put 16 of them
in 120 ml. of BVT in a small finger bowl giving a fluid depth of
about 1.5 cm. This culture received the same care as the Loomis
stock, except that no Hydra were discarded until the total reached
150 (on day 21). We saw the first sexual Hydra (a male) on day
10 when the population was 34. It was isolated for starting a
clone. In the next 25 days, as many as 15 males and 2 females were
seen at one time. On each of three days, one male was isolated. It
is the four male clones I wish to describe now. Each male was put
in a 30 ml. beaker as described for the isolation experiments but
the population was allowed to increase. When each population
leached 15 or 20 the clones were transferred to small finger bowls.
When the populations reached 150 they were kept constant by daily
random thinning. Two clones were aerated with room air; the other
two were partially covered and were not aerated.

Figure 4 shows the rhythmic nature of sexual differentiation in
the four clones. From 0-30 days (while the populations were small)
the ordinate values are the total numbers of sexual Hydra. From day
31 on, the ordinates represent percentages. Again, as in the cultures
of Loomis stock there was a rhythmicity in sexual difl^erentiation.
The four curves, on the basis of the number and height of the peaks,
can be roughly divided into two pairs: the triangle and circle, and
the square and inverted triangle. The triangle and circle show the
percentages of sexual Hydra in the cultures that were aerated with
room air; the other two show the percentages in the cultures that
were not aerated.

In order that you join me in a state of confusion, 1 want to re-
turn to the 16 Hydra culture from which these four clones were start-
ed. The culture is now 125 days old. It is not a clone; it presum-
ably contains both potential males and females. It has been sitting
on the laboratory bench partially covered; the population density

HELEN D. PARK

369

^ ^ 10 15 20 25 30 35 40 45 '50 55 60 65 70 '75 80 85 90 95 100
DAYS AFTER START OF EXPERIMENT

Fig. 4. Rhythmicity of sexual differentiation in four clones of Carolina
stock Hydra. 0-30 days, ordinates represent total numbers of sexual Hydra;
from day 31 on, ordinates represent percentages. • and A , aerated with
room air; ■ and ▼ not aerated.

has been the same as that of the clones (viz. 150); it has never
been aerated. The maximum number of sexual forms (7%) was
seen on day 34. Since then the culture has been almost free of sexual
Hydra; four, four and five were found on each of three days. There
have been long intervals (20 days) when no sexual forms could be
found.

In summary, then, under the conditions in our laboratory, the
Loomis stock Hydra, both in mass and isolation cultures, have un-
dergone alternating sexual and asexual periods during the past
three years. Furthermore, four clones of Carolina Biological Supply
Company stock have shown a similar periodicity for four months.
The original Carolina culture has remained relatively free of sexual
Hydra.

At present we cannot explain the alternating periods we have

370 THE BIOLOGY OF HYDRA : 1961

observed. In our current approaches we are considering both en-
vironmental factors, and factors which may reside in each hydra
itself.

REFERENCES

1. Brien, p. The fresh water hydra. Amer. Scientist. 48: 461-475.

2. Ito, T. 1952. Studies on the reproduction of hydra. III. Sexual periodicity

found in the hydra, Hydra magnipapiUata Ito. Memoirs Ehime Univ., Sect.
H (Sci.). 1: 221-230.

3. LooMis, W. F. and H. M. Lenhoff. 1956. Growth and sexual differentiation

of hydra in mass culture. /. Exp. Zool. 132: 555-574.

4. LooMis, W. F., 1957. Sexual differentiation in Hydra: Control by carbon dioxide

tension. Science 126: 735-739.

5. LooMis, W. F., 1959. In Cell, Organism and Milieu, XVII Growth Symposium.

The Ronald Press Company, New York, N. Y. pp. 253-294.

6. Park, H. D. 1959. Sexual cycles in Hydra. Anat. Rec. 134: 623.

7. Park, H. D., C. Mecca and A. Ortmeyer, 1961. Sexual differentiation in Hydra

in relation to population density. Nature (in press).

DISCUSSION

FULTON: Did you run all of the isolation cultures simultaneously?
Or when you were all through did you put them together as 200
days?

PARK: All of the isolation cultures in Figure 2 were run simul-
taneously. In Figure 4, I put all of the zero day clones back together.
As you can tell by the ends of the lines, we can calculate how
far apart in time they were started; I think not less than 3 days
nor more than 10 days.

FULTON: Do these fit together better if you put them in chrono-
logical time? I think it would be good to look in terms of periods in
the laboratory. In other words, what's happening on December 10th,
and so forth, as opposed to time in culture?

PARK: I understand what you're asking, and I can't say that I
know, because what we've seen is rather confusing. Sometimes they
fit better if they are put together chronologically. For instance,
if we take 10 Hydra plus another 10 which were run six months
later, plus another, and put them all back to zero, the periodicity of

HELEN D. PARK 371

the total disappears, this could be due to so many factors that I
wouldn't even want to speculate.

FULTON: I have had Carolina Hydra for about a year, and as far
as I know it is a strain of //. Uttoralis. Like the Looniis strain this
Hydra becomes sexual following the surface/volume ratio experi-
ments. That is to say, if you take the Carolina Hydra and put it in
a beaker and in a flat Petri dish (I did just the two extremes), it
will eventually become sexual in the beaker but not in the Petri
dish. The only thing is that it takes about a month. It's fairly
slow, but it does do it.

PARK: It is possible, of course, that the Carolina Hydra I received
are different from those Dr. Fulton received more than a year
earlier. Well, these are extremely interesting observations. Someday
I hope we will be able to fit our observations with those of Dr.
Fulton and Dr. Loomis.

LOOMIS: About seven years ago I asked Helen Forrest what made
hydra turn sexual. She answered that they seem to turn sexual in the
laboratory whenever they turned sexual in the neighboring ponds.
Now this, of course, seems silly, but if one is using Versene-treated
tap water, then it isn't as silly as all that. Tap water from a lake
varies in pCOo and other factors from one season to the next. It is
an interesting loophole in otherwise controlled experiments. It can
be surmounted of course by using BVC solution made from de-
ionized water. A Barnstead "red cap ' mixed resin cartridge removes
all COo from tap water, which is not true of the standard cartridge.

LYTLE: This is both a comment and a plea. In the interest-
ing report presented by Dr. Park, we have seen and heard evi-
dence of the existence of physiological differences among different
strains of a single morphological species of Hydra. I have recently
become concerned about this matter of subspecific differences in
hydroids since we now have evidence that such differences exist in
Hydra Uttoralis, Chlorohydra viridissima, Cordylophora lacustris,
and Craspedacusta sotverbii. No doubt in the next few years we will
see similar differences in many more species of hydroids. I have
just learned from Dr. Fulton at this symposium that he has observed
a number of rather striking differences among several strains of

2,72 THE BIOLOGY OF HYDRA : 1961

Cordtjlophora lacustris which he has isolated. In our laboratory we
have also found morphological and physiological differences
between different strains of Corclylophom lacustris and of Craspeda-
custa sowerbii which are both stable and transmissible. The extent
and significance of these differences are not yet fully analyzed, but
we are convinced that they do exist and that they are characteristic
of our various strains.

Therefore, in view of the growing complexity of this situation, I
should like to suggest that we establish some orderly and uniform
system for designating these various strains of hydroids. The in-
creasing number of investigators doing experimental work on hyd-
roids and our frequent exchanges of stocks made it imperative that
we take action on this matter.

There are already workable systems in operation for the iden-
tification of stocks of Protozoa, algae, Drosophila, and various other
organisms important in research. Perhaps we should model our
system after one of these existing schemes; at any rate, the impor-
tant thing is that we establish a system for the identification of our
stocks and that a central register be established for the purpose of
listing them.

PARK: Some of us have been talking about this since we arrived.
I don't know which laboratory could do it, but it seems to me
that we need a type of collection of coelenterate strains, or at least
of the ones that are being used in more than one laboratory.

Aging in Coelenterates

Bernard L. Strehler

Gerontology Branch, National Heart Institute, National Institutes of Health,
PHS, Department of Health, Education t~ Welfare, Bethesda, and the Baltimore
City Hospitals, Baltimore, Maryland.

Aging may be defined as the deerease in the funetional capacity
of an organism following its attainment of reproductive maturity
(27). According to this definition, aging is not a continuation of
development for aging generally expresses itself in a given species
as an increase in the probability of death, whereas de\'elopment
leads to increased functional capacity.

Different species of animals and plants age in different ways
(21). They age in accord with evolutionary forces, for length of
life, like other features of organisms, is an adaptation, at least in
part, to the niche which an organism occupies. Aging comparable to
that occurring in man and other metazoans probably makes its first
appearance in the coelenterates. There appeared to be controversy
for some time regarding the presence or absence of aging processes
in Hydrozoa, particularly in hydra ( 7 ) . Boecker ( 4 ) , Berninger
(2) and Hertwig (19) found that their cultures of hydra underwent
a depression with accompanying cytological changes. However,
Goetsch ( 15 ) improved culture conditions and kept individuals of
Pelmatohydm oligactis and another species alive for 27 months. He
believed that hydra, as well as Actinians, were capable of main-
taining themselves in status quo indefinitely. Gross (16), on the
other hand, failed to keep any individual of P. oligactis alive for
more than about a year and noted changes which he called "senile"
beginning at about the fourth month of life. Pearl and Miner (23)
used Hase's data (18) to construct a life table for hydra. David
(11) kept records of P. oligactis and was convinced that the in-
dividual animals tended to die between 20 and 28 months. However,

37.3

374 THE BIOLOGY OF HYDRA : 1961

Schlottke (25) made very careful cytological studies and, more-
over, suggested that David's histological sections were heavily para-
sitized. Schlottke's observations can be summarized as follows. There
appears to be an aging process in ectodermal cells which is char-
acterized by nuclear changes, e.g., pyknosis. He noted that the cells
move from the ectoderm into the endoderm after they degenerate
and observed the appearance of what he called "guanine deposits"
as the remains of cells which had been resorbed into the endoderm.
Schlottke also noted that degenerating nematocysts tended to move
into the endoderm.

Schlottke's early view is quite similar to that of Brien (5) who,
in 1953, published evidence, based upon marking experiments, that
there is a continual formation of new cells in the region around
the hypostome and that this is followed by a continual, slow (but
systematic ) movement of cells down over the surface of the column
of the hydra body to the foot where death and resorption take
place. One of the reasons that coelenterates are valuable in aging
studies arises from the fact that certain representatives of the phy-
lum make it evident that there is no necessity for senescence in
metazoa per se, just as certain immortal clones of protozoa demon-
strate (26) that sexual reproduction is not necessary for clonal
immortality.

A most charming description of a long-lived, and proba]:)ly
immortal coelenterate, was published by Ashworth and Annandale
in 1904 ( 1 ) : "We have, during the last two years, made a series
of observations upon specimens of Sagartia troglodytes (later re-
identified as Ceretis pediincitlatiis) which are at least 50 years old
and have thought it worthwhile to give a somewhat detailed account
of these. So far as we can ascertain, there is only one other recorded
case of longevity in coelenterates and very few in the whole of the
invertebrates. These specimens of Sagartia were collected by Miss
Ann Nelson (Mrs. George Brown) on the coast of Iran some few
years previous to 1862 (the exact date has not been recorded) and
were placed in bell jars containing sea water. In 1862, they were
transferred to the care of Miss Jessie Nelson, in whose possession
they still remain and to whom we are indebted for the opportunity
of observing these interesting anemones. Sixteen of the original
specimens are still living, so that they have lived in captivity for

BERNARD L. STREHLER 375

about 50 years. They are kept in a bell jar about 13 inches in dia-
meter and 9 inches in depth. The original specimens are all to-
gether on a piece of stone which bears a number of deep depressions
in which the anemones have ensconced themselves. These conditions
closely resemble those in which Sagartia troglodytes are usually
found, the specific name of this anemone being derived from its
favorite habit of dwelling in holes and crevices of the rocks. These
specimens have been under constant observation since 1862 and
there can be no doubt that they are the original ones."

These animals were later transferred to the Edinburgh Zoo and
lived until 1942 when all of them were simultaneously found dead
one morning (7). I doubt that they died of "old age."

It certainly seems well established that a process of clonal aging,
such as frequently occurs in protozoan cultures, is not a regular
process among coelenterates ( 20 ) . Lines of hydra in which the only
means of propagation was asexual budding have been kept for
decades without sexual crossing. In our own studies of Campamdaria
ftextiosa, a colonial hydroid, (30, 31), we have kept a clone growing
vigorously over the last three years on an artificial medium in the
laboratory without sexual crossing. This strain was obtained earlier
from Crowell (8, 9, 10) who had likewise kept it and perpetuated
it as a clone for a numlier of years.

Although, in the opinion of most recent investigators, certain
Hydrozoa such as hydra and probably many species of Anthozoa
do not undergo individual aging, there are closely related species
such as Obelia commissumlis and Campamdaria flexuosa which
do undergo a clear and most remarkable aging process. The details
of the senescence and death of Campamdaria hydranths is currently
being investigated in our laboratory. The developmental history of
clones of this species is approximately as follows. The animal grows
by sending out a root-like structure called a stolon which grows
on a hospitable substratum, either rock, piling or, even in some
cases, an algal surface (e. g., Fuciis). At periodic intervals, upright
branches appear as shoots from the main stolonic growth. These
proceed upwards for a certain distance, acquire a series of annula-
tions, the most distal of which eventually enlarges into a bulblike
structure. This primitive structure then elongates, acquires a rhyth-
mic muscular contractility, lays down a protective covering shield

376 THE BIOLOGY OF HYDRA : 1961

or chitinous perisarc, develops tentacles at the upper end, hollows
out and finally perforates a mouth in the center of the tentacles
(see Fig. 1). For 4 or 5 days, this hydra-like animal, growing
on a branched stalk, catches Crustacea or other suitable prey with
the batteries of nematocysts in its tentacles, ingests them, dissolves
their contents which are taken up by a phagocytic process into the
endodermal cell layer or transmitted back down along the branching
root-like stolon to other individuals in the colony or to the region
of apical growth.

tk \r . «k f r

V. V V V

J K

Fig. 1. Development of Campanularia hydranth. (Taken from time lapse
sequence).

I would like to suggest that these contrasting species of coelenter-
ates are useful in studies of the biological basis of senescence be-
cause they furnish us with exaggerated models of parallel systems
we may observe within more highly evolved metazoa such as human

Fig. 2. Section of thick human skin showing sequence of cell growth
(A), Differentiation (B, C), Death (D, E, F). A — germinal zone; B — prickle
cell zone; C — zone of RNA granules (Granulosa); D — area of lysis (note
nuclei in process of solution); E — area of cytoplasmic dissolution; F — kera-
tinized zone.

377

378

THE BIOLOGY OF HYDRA : 1961

beings. One such analogy is illustrated in Figure 2 which shows a
section of thick human skin. You will notice that there is a generative
zone, in which cell division and growth take place, and next to it the
so-called prickle cells in the spinosa in the process of differentiating.
Further toward the surface we see the granular layer. In addition
to showing a strong basophilia, due to the presence of acidic sub-
stances which have been identified as RNA by Leuchtenberg (22),
we observe in cells just distal to the granulated cells the complete
disappearance of these acidic substances. I believe it is likely that
this lysis is due to the action of lysosomes, bodies which DeDuve
(12) identified some years ago as contaminants in mitochondrial
fractions. Such structures occur in many cell types and, in the event
that the cells are damaged, they are activated to break down cellular
contents and thus clear the way for repair processes. In this present
case, they appear to hydrolyze all of the cell contents except, pre-
sumably, keratin and a few other substances.

Fig. 3. Photomicrograph of old human myocardium (86 years old) taken
by its own fluorescence in U.V. light. Bright spots are lipofuscin granules.
400X magnification.

BERNARD L. STREHLER 379

The opposite extreme in cell types is illustrated in Figure 3
which shows a section from old human myocardium. These cells,
in contrast to skin cells, do not die regularly, but rather live for the
lifetime of the animal. A most interesting feature of such cells is the
fact that they accumulate a fluorescent brown pigment known as
lipofuscin ( 17 ) . We are attempting to isolate and characterize this
substance. The fluorescent component appears to be an auto-
oxidized, unsaturated lipid. We have shown that this material accu-
mulates linearly with time (32), at the rate of about three-tenths
percent of the total heart volume per decade. Gedigk and Bontke
( 14 ) have demonstrated that these granules possess a number of lytic
enzymes and may thus be a type of lysosome. Lipofuscin apparently
accumulates in all non-dividing cell lines. On the other hand, it
does not generally appear in dividing cell lines. I would like to
suggest that the heart is analogous to Campamdaria and that there
exists an analogy between anemones or hydra and the regularly
replenishing structure, skin. In the former case, there is no regular
cell replacement since the cells of the nervous system and the heart
are carried through the life of the individual. On the other hand,
the skin is in a continual process of replacement just as is hydra
with its growth in the hypostomal region and the death of cells at
the base and probably at the ends of the tentacles.

In support of this thesis, I would like to concentrate on a com-
parison between certain histochemical properties of H. littoralis and
Cajnpannhria which are being studied in cooperation with Dr.
Mary Anne Brock (6). In Figure 4 is illustrated the regression
process in Campamdaria as recorded in time lapse movies. Campa-
nularia exhibits regular peristalsis very much as does Cordylor-
phora. The histological appearance of a young Campanularia is
shown in Figure 5.

The first sign of the senescence of the individual hydranth is a
slight shortening of the tentacles and the appearance of knobbiness
accompanied by a change in refractive index on the end of them.
The tentacles then begin slowly to shorten and draw in toward
their bases. After the tentacles have contracted completely, there
is a sudden release of something which breaks down the intercellular
cement and, at the same time, results in cell autolysis. A hydranth
in this stage is shown in Figure 6. Finally, the entire contents of the

380

THE BIOLOGY OF HYDRA : 1961

Fig. 4. Regression of Campanularia hydranth. (Taken from time lapse
sequence). Note thickening of tentacles prior to resorption. Entire sequence
takes about 6 hours.

dead animal is passed l^ack into the colony from whence it came,
and all that is left as a reminder of the former inhabitant is the
empty hydrotheca.

What is the mechanism of cell death underlying these changes?
Is it similar to that which has been postulated or shown in other

BERNARD L. STREHLER

381

Fig. 5. Normal young Componu/or/o section. Magnification 500X.

Fig. 6. Regressing Campanularia hydranth. Magnification^SOOX.

species? Might it be the activation of lysosomes? In order to test this
thesis, we have compared H. littoralis of the Loomis strain with
Campanularia for the presence and distribution of acid phosphatase
f>ositive granules. This is one of the simplest ways to localize pre-
sumptive lysosomes (13). Acid phosphatase is one of the enzymes
which lysosomes generally contain.

Figure 7 illustrates a young Campanularia, stained for acid

Fig. 7. One day old Campanularia hydranth. Gomori acid phosphatase
stain. Note strong nuclear stain and essential absence of strongly positive
small particles. Magnification=about 900X. (From Brock and Strehler, un-
published).

Fig. 8. Ten day old regressing hydranth of Campanularia. Gomori acid
phosphatase stain. Both nuclei and a multitude of small cytoplasmic granules
give positive stain. 500X magnification. (From Brock and Strehler, un-
published).

.382

BERNARD L. STREHLER

383

phosphatase by Gomori's method (24). Note that there is practi-
cally no acid phosphatase except in the nuclei, although there is an
occasional granule here and there.

Compare the sparseness of acid phosphatase in the young ani-
mal with Figure 8 which shows a ten-day-old hydranth in early
stages of regression. Particularly notice the enormous numbers of
very small acid phosphatase-positive granules, which are nearly
everywhere in the gastrodermal cells. Notice that the tentacles have

Fig. 9. Electron micrograph of regressing hydranth. Note cytoplasmic
disorganization, vacuolization. Magnification about 24,000X. Taken in
collaboration with Dr. D. Brandes, Pathology Department, Baltimore City
Hospitals.

384

THE BIOLOGY OF HYDRA : 1961

contracted somewhat. The huge vacuoles in the gastrodermal cells
are diminished or absent. These acid phosphatase positive granules
are of very uniform size.

Figure 9 is an electron micrograph of a regressing hydranth
(taken in collaboration with Dr. David Brandes of the Pathology
Department, Baltimore City Hospitals) and shows the complete
intracellular disorganization which takes place during this process.
An occasional mitochondrion still seems to have a few cristae intact
but the high degree of vacuolization and lamination in this section
is completely foreign to the normal anatomy of this organism.

Fig. 10. Accumulation of acid phosphatase-positive granules in the tips
of the tentacles of Hydra. Gomori stain. 500X. (From Brock and Strehler, un-
published).

For comparison in the succeeding figures, evidence of acid
phosphatase activity in certain interesting regions of Hydra is pre-
sented. Note the gradient of increasing acid phosphatase activity
as one moves toward the tips of the tentacles. This activity is con-
fined to the gastrodermal cells of the tentacle tips (Fig. 10).

In the base, one also finds acid phosphatase activity in the pre-

BERNARD L. STREHLER 385

cise area in which one would expect it (see Fig. 11). The enzymes
(and lysosomes?) are locahzed in a pedal disc, although there is
some accumulation even in cells that presumably are differentiating
into lysosome-containing cells higher on the column. I think this
would be interesting to study — the kinds of structures which con-
tain the enzyme and whether they are similar to those which are
present in other animals. Notice that acid phosphatase occurs both
in the gastrodermal and ectodermal layer.

We were interested to see whether there are changes in the
numbers of similar granules in Campanidaria, particularly in the
tentacular region where regression starts. In the young hydranth,
there is very little acid phosphatase activity. By contrast, there are
enormous numbers of very uniformly sized granules in the 10-day-
old hydranth as was shown in Figure 8. Note the deposition of acid
phosphatase positive material along the cytoplasmic septa separat-
ing the gastrodermal cells in the tentacles of a 9-day-old hydranth
shown in Figure 12. These cells have huge vacuoles with an om-
mentum-like cytoplasmic extension containing the nucleus hanging

Fig. 11. Accumulation of acid phosphatase-positive granules in the pedal
disc of Hydra. Gomori stain. 500X magnification. (From Brock and Strehler,
unpublished).

Fig. 12. Acid phosphatase-positive granules (arrows) in the tentacles of
an 8 day old Campanularla. 1000X magnification. Gomori stain. (From Brock
and Strehler, unpublished).

3S6

BERNARD L. STREHLER

387

into them. After seeing the distribution of the acid phosphatase
granules, we re-examined some electron micrographs that we had
taken. We plan a much more systematic study of the degenerating
hydranths to see whether they contain structures reminescent of
lysosomes. In Figure 13, which illustrates a group of tentacles
at their point of attachment to the body wall, are some objects
which may be suitable candidates. They are certainly not mitochon-
dria which are also located in these very thin walls.

Now, what can one say about the functional capacity of old

^irT

Fig. 13. Electron micrograph of a Campularia tentacle. Arrows indicate
possible loci of acid phosphatase activity. Magnification=about 4000X.
(From Strehler and Brandes, unpublished).

388 THE BIOLOGY OF HYDRA : 1961

TABLE 1

Food catching ability vs. Age
Series I (about 2.0 Artemia/hydranth — fresh sea water)

Hydranth age (days)
No. of hydranths
No. of Artemia caught
Artemia caught/hydranth

1
10

13
1.3

1.4

1.35

0.93

1.33

Series II (about

0.5 ArtemJo/hydi

ranth-

— c

irtificial sea

wate

!r)

Hydranth age ( days )
No. of hydranths
No. of Artemia caught
Artemia caught/hydranth

299

.24

224

.21

3
187

47
.25

150

.21

104

.49

63
21
.33

7
35

6
.17

.,30

TABLE 2
Ingestion time vs. Hydranth age

Age of hydranths 0 12 3 4

No. of hydranths tested 31 26 52 18 16

Average time for ingestion in seconds 257 256 300 295 237

Campamilaria? How do they differ from young ones? Dr. Crowell
(who did much of the basic work upon which this study is based)
and I have measured a number of physiological capacities vs. age

Fig. 14. Photomicrograph of Cam-
panular'ia ingesting Artemia labeled
with fluorescent dyes (0.1 % acrifla-
vine). From a time lapse sequence. Note
appearance of fluorescent digest in
upright proximal to upper hydranth.

BERNARD L. STREHLER 389

(31). Some of these are shown in Tables 1 and 2. One of the things
that we measured was the efficiency of catching Artemia. This did
not seem to be altered between one and five days of age, which is
about the greatest longevity of appreciable numbers of hydranths in
Woods Hole at 17-18 . Similarly, the digestion time was measured
by feeding fluorescent labeled Aiiemia to Camponularia and then
measuring the time required for the first fluorescent digest to appear
in the region proximal to the hydranth (see Fig. 14). As measured
in this way, no differences between young and old were observable.
Neither did the egestion time nor the maximum number of Artemia
the hydranths can consume change with age.

In short, the only striking differences we have found between
young and old CampanuJaria, other than the above-mentioned acid

TABLE 3

ATP content/hydranth vs. age

Stage ATP/hydranth = (g X 10'")

Hydranth bud 15.0

Early differentiation 7.0

Complete differentiation but not extended 33.0

1 day old (young) 18.0

2-3 days old (middle-aged) 12.0

4-5 days old (old) 4.5

phosphatase accumulation, is a difference in the level of the adeno-
sine triphosphate as measured by our firefly enzyme ATP assay
method. We observed a decrease to about one-third of the total
ATP level in passing from one to five days of age (see Table 3).
This value was calculated per hydranth rather than on a dry weight
basis and we do not know whether there is a change in the dry
weight of Campainiknia during this time interval. We can thus not
say whether this ATP concentration drop is due to a decrease in
the intracellular concentration or volume.

Complete lack of oxygen for a period of several hours does not
produce degeneration of Campanidaria, although we have obtained
evidence from time lapse studies that partial anaerobiosis can rapidly
induce degeneration. During the complete absence of oxygen for

390

THE BIOLOGY OF HYDRA : 1961

"MI'l'I'I'liMIM'I'Ml'IMMMMilMMiIim'PIM'I'IMilMil'MMI'MI'lM'I'

LiJ
CD

|ii'"nn

—nil ^ -n J 1

|iiT|'r|i|i[Tp|i|in'iF''Tniini|inl

1 1 1 1 ,] F] n ' 1 1 1 '"i"i n M h MTM ' 1 1 1 'Ti'i'i 1 1 1 [? 1 1 1' n ' I t'th i i H' i i i"i|j i ' fvv i '

2468 2468 2468 2468 2468 2468 2468 2468

CONTROL 500R 2 KR 5KR 25KR 50KR lOOKR 200KR

DAYS OF AGE

Fig. 15. Distribution of ages at death of Campanularia hydranths of
various ages after exposure to various dosages of X-rays. The highest peak
represents 23 hydranths.

the two hour period which we used, it may be that there is a com-
plete arrest of metabohc activity inckiding the synthesis of the lytic
enzymes.

It was current doctrine about two years ago that high energy
radiation is analogous to time in its effect on aging. We therefore
undertook to see whether Campanularia is aged by high doses of
radiation. We gave dosages up to 200,000 r and then followed the
longevity of individual hydranths. Figure 15 shows the average life
time at various dosages. The mean life time, under the control
conditions, was about 2.7 days, whereas at 100,000 r the average
life time was about 6.3 days. This radiation dose more than doubles
the longevity of the hydranth!

Another remarkable fact is that hydranths continue to differen-
tiate and be initiated even one week after exposure although the
colony eventually dies. Figure 16 shows the survival curves of these

BERNARD L. STREHLER

391

Fig. 16. Survivors (% remaining alive) at various ages following X-ray
exposure.

animals plotted on a linear scale while Figures 17a and b show a
control and irradiated colony at various times after exposure.

What relationship does aging in Campamilaria have to aging in
general? First, I believe it will turn out that the mechanism of cell

392

THE BIOLOGY OF HYDRA : 1961

Fig. 17. Time lapse photographs of control (upper) and irradiated (lower^
colonies of Campanularia — 1, 36, 72, 108, 144, and 180 hours after receiv-
ing 100,000 r.

death in Campanularia and in hydra is probably quite similar to
that which occurs in humans. Second, it is evident that mortality is
an evolved character that may or may not express itself even in
closely related species. Why should one strain of hydrozoan be

BERNARD L. STREHLER 393

essentially immortal and why should another strain be so highly
mortal? In searching around for an answer to this, Dr. Crowell
and I, in the absence of clear-cut functional differences between
the young and old indi\iduals, settled upon an interpretation which
incorporates certain of his earlier studies on the response of the
colony to restricted feeding ( 9 ) . He noted, as he has mentioned at
this meeting, that the individuals who have precedence in such a
colony as Campanularia are those that are at the top of an upright,
and that the lateral growth of the stolon and the growth of the
apical hydranth are not so readily inhibited. This suggested to us
that the colony distributes its feeding individuals on the periphery
as a sort of umbrella during periods of poor food supply. They
thus are in a position to intercept the greatest number of prey — an
economically efficient distribution of a limited supply of protoplasm.
Since these animals live under conditions of quite variable food
supply, we postulated that their senescence is a built-in clock that
forces the colony as a whole to evaluate on a very regular schedule
the adequacy of its food supply. If the food supply is not adequate,
then regeneration in the lower parts of the uprights does not take
place. If there is a large amount of food available, then it is economi-
cally feasible to regenerate hydranths all up and down the upright
and thus to survi\ e.

In general terms, a paradox is apparent; namely, only those
animals which have devised a means of replacing all of their cells
on a regular schedule are able to live as individuals for indefinite
periods. Part of this process of replacement involves, of necessity,
a destruction of cells in a systematic, ordered way ( 28 ) . If it takes
place at the boundary of an animal or in a linear, ordered pro-
gression of some sort, then the animal, pro\ided it has a germinal
core of cells, is capable of continuing to exist indefinitely in a steady
state. If it has no capacity for replacing its cells, or its cell parts, but
rather accumulates damage, noxious substances or accidental by-
products of metabolism, then it will eventually die. Hydra and the
anemones are probably immortal because they have devised and
maintained evolutionarily a systematic replacement scheme. We and
Campanularia are mortal either because it is advantageous to the
species' survival to be mortal or, either directly or as a by-product
(3, 33) of some other advantageous genetic character, as appears

394 THE BIOLOGY OF HYDRA : 1961

more likely in our case, because selection pressure has not been
sufficiently severe to provide a replacement regimen for those cells
and tissues which are relativel)' well shielded from accidental dam-
age or loss (29).

ACKNOWLEDGEMENTS

The author wishes to acknowledge the collaboration of Drs.
David Brandes and Mary Ann Brock in many of these studies as
well as the constant and invaluable assistance in all phases of this
work bv Mr. Malcolm Gee.

REFERENCES

1. AsHWORTH, J. H., and N. Annandale. 1904. Observations on some aged speci-

mens of Sagartia troglodytes and on the duration of life in Coelenterates.
Proc. Roy. Soc. Edin., 25: 295.

2. Berninger, J. 1910. Uber Einwirkung des Hungers auf Hydra. Zoo/. Anz., 36:

271-279.

3. Bidder, G. P. 1925. The mortaUty of plaice. Nature, London, 115: 495

4. BoECKER, E. 1914. Depression und Missbildungen bei Hydra. Zoo/. A)iz., 44:

75-80.

5. Brien, p. 1953. La Perennite Somatique. Biol. Rev., 28: 308-349.

6. Brock, M. A., and B. L. Strehler. Unpublished.

7. Comfort, A. 1956. The biology of senescence. Rinehard & Co., New York,

p. 257.

8. Crowell, S. 1953. The regression-replacement cycle of hydranths of Ohelia

and Campamdaria. Physiol. Zool., 26: 319-327.

9. Crowell, S. 1957. Differential responses of growth zones to nutritive level, age

and temperature in the colonial hydroid, Companularia. J. Exp. Zool, 134:
63-90.

10. Crowell, S., and C. Wyttenbach. 1957. Factors affecting terminal growth in

the hydroid, Campamdaria. Biol. Bull, 113: 233-244.

11. DAvm, K. 1925. Zur Frage der potentiellen Unsterblichkeit der Metazoen. Zool.

Anz., 64: 126.

12. De Duve, C. 1957. The enzymatic heterogeneity of cell fractions isolated b\-

differential centrifugation. Symp. Soc. Exp. Biol., 10: 50-61.

13. Essner, E., and A. Novikoff. 1960. Human hepatocellular pigments and lyso-

somes. /. Ultrastruc. Res., 3: 374-391.

14. Gedigk, p., and E,. Bontke. 1956. Uber den Nachweis von hydrolytischen

Enzymen in Lipopigmenten. Z. Zellforsch., 44: 495-518.

15. Goetsch, W. 1922. Lebensdauer und Geschlechtige Fortpflauzung bei Hydra.

Biol. Zhl, 42: 231.

16. Gross, J. 1925. Versuche und Beobachtungen liber die Biologic der Hydriden.

Biol. Zhl, 45: 385-417.

BERNARD L. STREHLER 395

17. Hamperl, H. 1934. Die Flunrescenzmikoskopie Menschlicher, Gevvebe. Virchows

Arch., 292: 1-51.
1(S. Hase, a. 1909. tjber die deutschen Su.sswasser-polypen Hydra ftisca. Arch, fiir

Rossci^-und Gessclhchaftshioh)gie, 6: 721-753.

19. Hertwig, R. 1906. Uher Knospung und Geschlectentwicklung von Hydra fiisca.

Biol. Z/;/., 26: 489-508.

20. HixLEY, J. B., and G. R. De Beer. 1923. Studies in dedifferentiation. IV.

Re.sorption and ditterential inliiliition in Obclia and Campanidaria. Quart.
]. micr. Sci., 67: 473.

21. KoMGSBERG, I. R. 1960. On the relationship between development and aging.

Newsletter (Geront. Sac), 7: (3), 33-34.

22. Leuchtenherger, C, and H. Z. Lund. 1951. The chemical nature of the

so-called keratohyaline granules of the stratmn granulosuni of the skin.
Exp. cell Res., 2: 150.

23. Pearl, R., and J. R. Miner. 1935. Experimental studies in the duration of life.

XIV. The comparative mortalitx of certain lower organisms. Quart. Rev.
Biol., 10: 60.

24. Pe.\rse, a. G. E. 1960. His-tocJiemistry. theoretical and applied. Little, Brown

& Co., Boston, 2nd Ed., 998 pp.

25. ScHLOTTKE, E. 1930. Zellstudien an Hydra. I. Alteni mid abbau von Zellen mid

Kernen. Z. Mikr. Anat. Forsch., '22: 493-532.

26. SoNNEBORN, T. M. 1960. Enormous differences in length of life of closely related

ciliates and their significance. In: B. L. Strehler et al. (Editors), The
Biology of Aging. Amer. Inst. Biol. Sci., Washington, Pub. No. 6, p. 289.

27. Strehler, B. L. 1959. Origin and comparison of the effects of time and high-

energy radiations on living systems. Quart. Rev. Biol., 34: 117-142.

28. Strehler, B. L. 1960. Dynamic theories of aging. In: N. W. Shock (Editor),

Aging—Some Social and Biological Aspects. Amer. Assoc. Ad\ . Sci., Wash-
ington, Pub. No. 65, pp. 273-303.

29. Strehler, B. L. Time, cells and aging. Academic Press, New York, (in press).

30. Strehler, B. L. Unpublished.

31. Strehler, B. L., and S. Crowell. 1961. Studies on comparative physiology of

aging. I. Function vs. age of Campanularia jiexuosa. Gerontologia, 5: 1-8.

32. Strehler, B. L., D. D. Mark, A. S. Mildvan, and Malcolm Gee. 1959. Rate

and magnitude of age pigment accumulation in the human myocardimn.
/. Geront., 14: 430-439.

33. \A'iLLiAMS, G. C. 1957. Pleiotropy, natural selection and the evolution of senes-

cence. Evolution, 11: 398-411.

DISCUSSION

MARTIN: By 100,000 r, do you mean tissue dose or dispensing
dose?

STREHLER: Tissue dose.

MARTIN: You measure it underneath the water?

STREHLER: It was calculated for the chamber in which it was

396 THE BIOLOGY OF HYDRA : 1961

irradiated. Since it was a high energy photon I beUeve it was not
attenuated much by the water.

GOREAU: Are there other criteria for measuring age? Two years
ago we measured the effect of size on specific calcification rates on
a free hving coral where size was an indication of age. In this
particular species, Manicina arcolata, we found a progressive reduc-
tion in the calcification rate (per mg. of protein nitrogen) as the
colonies got larger. The difference between the smallest (50 mg. )
and the largest ( 149 g. ) colonies tested was almost two orders of
magnitude (Goreau, T., and N. Goreau. 1960. Biol. Bull 118:419).
Now, it is interesting that these particular corals hardly ever grow
beyond 500 grams. This may have some ecological significance
for if the individuals get much larger and heavier than that they
may sink into the sediments since most of them are not attached.
However, many other species of corals do not show such a regulated
growth pattern. I suspect that some species grow indefinitely.

STREHLER: The maximum ages for corals which are quoted
in Comfort's excellent book (which I recommend to those of you
who might be curious about senescence) is only about 28 or 30
years. Now this is based upon size estimates. 1 would be delighted
to see somebody try to find a better index of age than simply size.

GOREAU: On the basis of some of the accretion data which I
referred to in my talk on Thursday, I calculated that corals weigh-
ing about 200 tons may be as much as 800 years old.

STREHLER: You think these come from a single individual?

GOREAU: Not necessarily, but the specimens used in our experi-
ments were clones descended from single planulae.

STREHLER: The difficulty here is that this is more like a hydra
clone, it seems to me, than an individual animal.

GOREAU: Some corals, like the branching species, have an in-
determinate growth pattern and can probably be considered im-
mortal, because they can grow as long as there is room. This is
probably not true of the massive ones in which the skeletal mass
would increase much faster than surface area. These would col-

BERNARD L. STREHLER

39?

Fig. 1. Sketches to illustrate Nathanson's experiment. A. An isolated
hydranth. B. The hydranth may produce a short stolon in a few hours. C.
When regression of the hydranth occurs the stolon grows. D. A new upright
has started to form; regression of the old hydranth is nearly complete. E. A
new hydranth is produced.

lapse or become buried under the increased weight. However that
may be, I beheve all reef corals are clones.

STREHLER: Studies on the incorporation of tritiated thymidine
into the DNA of Metridium or of other long-lived forms would be
of interest in outling the pattern of cell division and replacement.

CROW ELL: May I go back to what we were talking about this
morning? How, and in what form, are materials moved to regions
of growth? These sketches, Figure 1, show the results obtained by
Nathanson (Nathanson, D. L. 1955. The relationship of regener-
ative ability to the regression of hydranths of Campamdaria. BioL
Bull. 109: 350). He cut off single hydranths of Campanularia, placed
them in stender dishes, but did not feed them. A hydranth merely
sat for from one to four days. In some cases it produced a little bit
of stolon, as shown in Figure IB. When a hydranth began to regress
the stolon elongated, as shown in C and D, then it sent up a new

398 THE BIOLOGY OF HYDRA : 1961

upright and produced a new little liydranth, Figure IE. Presumably
all of the new growth is made possible by utilizing the debris of
the regressing hydranth and just a small quantity of good, new cells
which at first were assembled at the base of the isolated hydranth.
If so, this experiment of Nathanson points to a very rapid utilization
and reorganization of this stuff in the building of new cells. No one
has looked at such preparations with anything more powerful than
a binocular microscope. This looks like a good place to make a
start on the problem.

CHAPMAN: Do I understand you to indicate that you thought
the dense particles in your electron micrographs indicated by the
arrows were lysosome particles?

STREHLER: Possibly.

CHAPMAN: If you thought they were, I want to tell you I thought
they weren't for two reasons. One, it seems to me they are too large
to be lysosomes. Lysosomes fall in the range between the smallest
mitochondria and the largest elements of the so-called microsome
fraction. And two, they looked almost homogeneous to me and I
would expect a more dense outer shell.

STREHLER: I think you are in error because lysosomes are quite
variable in size. If you look at Essner and Novikoff's electron
photomicrographs of liver lysosomes, the size variation occurs and
they frequently appear with a moon shaped heavy border. This
looks like a vesicle in this micrograph. But the acid phosphatase ac-
tivity is actually usually associated with the dense staining portion.
I don't think there is really enough structure in those particles to
answer the question.

Studies on Chemical Inhibition
of Regeneration in Hydra

Robert E. Eakin

Clayton Foundation Biochemical Institute and the Department of Chemistry,
The University of Texas, Austin, Texas.

First, I would like to express my disappointment that Dr. Ham
and Dr. Spangenberg, former students of mine, were unable to get
to the meeting to present their own contributions. They were re-
sponsible for developing the research program I shall discuss.

I would like to summarize the results of three phases of our
investigations: first, factors influencing the regenerative response of
hydra under normal conditions; second, the effects of chemical
agents upon regenerative processes; and third, some biochemical
observations on the effect of an agent which uniquely arrests regen-
eration — lipoic acid. (A fourth phase, histological studies on both
normal and treated organisms, is included on pp. 4L3-423 of this
volume. )

FACTORS INFLUENCING REGENERATION OF
UNTREATED HYDRA

Seven years ago I would not have anticipated I would be at this
late date reporting on the control of factors influencing normal
regeneration. But we've had our troubles.

When Dr. Ham originally initiated the use of hydra in our
laboratory, he did a thorough and comprehensive study on the
environmental factors which influence the regeneration rate of
H. littoralis (4). But over a period of several years' time, we en-
countered a considerable degree of inconsistency in the behavior of

3.9.9

400 THE BIOLOGY OF HYDRA : 1691

our stock clone when compared to previous responses which the
organisms had given. Also, we were continually confronted with
daily erratic behavior in Hydra taken from the same culture dish
and treated in what we believed to be an identical fashion. And
finally, it was found that our stock clones were uniformly parasitized
by an intracellular protozoan, a species of Microsporidia. It was this
point that we were ready to throw all our clones back into the lily
pond from whence their great grandparent had come. In our earlier
studies, we used one strain of Hydra and indeed were apprehensive
about bringing into the laboratory any other strain of Hydra littor-
alis, lest there be mixing of stock clones. However, because of our
difficulties we did bring other strains of //. littoralis into our labora-
tory — retreated, as it were, in our program— to re-examine more
critically factors which could be causing variations in the response
of "normal" untreated hydra.

Although it was a laborious undertaking, it pro\'ed worthwhile
in that it not only pointed out the causes of the inconsistent results
we had been getting, but also increased by a considerable extent
our knowledge concerning both extrinsic and intrinsic factors
which influence regeneration. This new data, in turn, led to histologi-
cal studies giving valuable information concerning some of the basic
factors influencing the rate and extent of regeneration and enabling
IIS to develop a hypothesis for use in planning future investigations.

For the purpose of discussion, the factors studied can be classi-
fied as (A) intrinsic factors and (B) extrinsic factors.

In selecting criteria for measuring regenerative capacities of
hydra, we have used two measurements which can be rapidly
determined on a large number of organisms — namely, (a) the
length: width ratio observed for the longest developing tentacle
formed during the early stages (18th to 24th hour) of regeneration,
and ( b ) the total number of tentacles observed after the regenerative
process is well along ( after the 44th hour ) . The early measurement
of the extent of the growth of the future tentacle gives some indica-
tion of the rate at which morphological processes can be initiated.
The total number of tentacles regenerated expresses roughly the
total amount of morphogenic change. These two determinations ob-
viously are a measure of the composite effects of many individual
factors, but they have permitted us to run screening tests on thou-

ROBERT E. EAKIN

401

sands of organisms for the gross effects of a number of extrinsic
and intrinsic factors which affect development. For example, infor-
mation which we gained from later histological studies enables us
now to look at gross regeneration data and make some educated
guesses of interstitial cell patterns. Our ultimate goal is to relate
the effect of physiological and chemical agents to much more
specific phenomena — namely, changes induced in biochemical and
structural patterns during the regenerative process, such as those
described in the histological and enzymatic investigations I shall
mention presently.

One intrinsic factor — the genetic differences in strains — is
illustrated in the left half of the first figure ( Table 1 ) which shows
the differences in the gross macroscopic responses that have been
studied in detail in seven strains of hydra (representing three
different species), the experimental procedures being those reported
in our previous publications ( 2, 3, 4) .

Htjdra littoralis — Strain I: These were Hydra derived from the
clone initially used in this laboratory ( 1 ) and provisionally identified

TABLE 1
Effect of site of severance upon rate of regeneration

Hypostomal cut

Mid-stomach cut

Extent o

f Regeneration

Extent of

Regeneration

Hours

TX"

Hours

TN»

Organism

22 24

units* "

units""

Chlorohydra

viridissima

3.4

— -

.... 6.1

2.6

3.3

.... 7.1

( green )

Chlorohydra

viridissima

3.0

_...

.... 6.0

2.1

2.8

.... 7.0

( brown )

Strain I

1.8

2.6

3.2 ..._

.... 6.3

.60

1.2

1.9 2.3

.... 6.6

Strain II

.81

1.6

2.5 ____

.... 4.5

.09

.14

.26 .57

.89 3.0

Strain III

1.6

2.1

2.4 -_._

.... 1.2

.22

.24

.28 ....

.41 .33

Strain IV

1.4

2.5

3.0 .-

.... 6.2

.00

.09 .22

.40 5.4

Hydra

oligactis

.70

1.6

2.7 .___

.... 5.5

.00

.00 .00

.11 1.4

Each value represents the average of 36 repHcates.

"Tentacle number at 48 hours.

"" Length :widtli ratio of the longest tentacle.

402 THE BIOLOGY OF HYDRA : 1961

as H. littoralis. All organisms in the clone were found to be infected
with Microsporidia, the parasitization occurring in both the epider-
mal and gastrodermal cells. Treatment of the Hydra with a fungi-
cide, Fumidil B, apparently eradicated the parasite as clones of
the treated Hydra have remained parasite-free for two years. These
Microsporidia have been identified tentatively through the courtesy
of Dr. R. R. Kudo as a species of Plistophora — a type of protozoal
parasite causing fatal infections of silkworms and honeybees. Re-
ported for the first time in Hydra, the infection in this organism is
not fatal, nor does it interfere with normal asexual reproduction.
Because of this, parasitized Hydra may provide a unique system
for the study of the life cycle of Microsporidia (7).

Hydra littoralis — Strain II: These were from a clone generously
furnished ])y Dr. Loomis and identified as H. littoralis.

Hydra littoralis — Strain III: In an effort to obtain a non-
parasitized Hydra closely resembling Strain I, a sexual cross was
made between an infected male of Strain I and a female of Strain
II. The resulting offspring were parasite-free. These clones have
been designated Strain III. (When the opposite cross was made,
the eggs hatched only rarely and the offspring were infected. )

Hydra littoralis — Strain IV: Another clone, referred to as
Strain IV, was developed from a Hydra found in a pond on the
University of Texas campus and has been tentatively identified
through the courtesy of Dr. L. H. Hyman as H. littoralis.

The two strains (I and IV) only tentatively identified as H.
littoralis have been considered as members of this species in view
of their close morphological resemblance to the positively identi-
fied Strain II and because of the readiness with which Strains I and
IV cross sexually with Strain II to produce \'iable offspring which
later become sexually reproductive. Although all four strains ap-
pear to be Hydra littoralis, there is variation in size, rate of regenera-
tion, and physiological responses. Strains I, II, and III are all very
much alike in appearance but Strain IV is a larger and a more
slowly moving Hydra.

Hydra oligactis: These are from clones developed from an or-
ganism purchased from General Biological Supply Co., Chicago, 111.

Chlorohydra viridissima (Green): This culture of Chlorohy-
dra was derived from specimens found in a local pond.

ROBERT E. EAKIN 4DS

Chlorohydm viridissima (Non-green): The non-green Chloro-
hijdra were obtained by depriving green organisms of light for
several weeks. Upon exposure to light, some soon regained their
green color but the others, although subsequently grown in the
light, have not regained their color after three years.

Not shown in this table is the growth rate by asexual budding.
This can be most easily expressed in the time required for a doub-
ling of the number of hydranths in optimally nourished clones.
The fastest growing hydra are the Cldoiohydra, both the green
and the non-green strains doubling in about 1.3 days. H. oligactis
and Strains I, II, and III of H. Uttoralis double in number in 2.0

TABLE 2
Regenerative response of a "typical" and an "atypical" subclone of Strain II

Age of subclones Tentacle number at 48 hours*

^'^y^ Typical Atypical

0 48 LS

24 1.2

53 4.8

57 1.4

68 2.0

12.3 1.6

151 4.8

168 2.7

188 1.6

"Average of 18 replicates.

to 2.4 days, but Strain IV is an unusually slower grower, having
a doubling time exceeding 6 days. There is no apparent correla-
tion between rate of asexual budding and the rate of tentacle
regeneration in the different strains.

A second intrinsic factor influencing regeneration — inherita-
ble variations arising within a clone — is illustrated in Table 2.
Some of our erratic behavior finally was traced to the "area-of-
the-dish" effect, that is, the regenerative response was related to
the area of the culture dish from which an organism was taken.
In determining the cause of this behavior, l3uds from parents used
in tests were subcloned and their subsequent behavior determined.
It was found that (a) some of the parent hydra regenerated few-

404 THE BIOLOGY OF HYDRA : 1961

er tentacles than others at 48 hours and that (b) the offspring
in the subclones showed the same characteristics as their parents.
Subclones selected on the basis of parents regenerating within 48
hours a normal number of tentacles — four to six — being desig-
nated "Typical" and those selected on the basis of parents regen-
erating fewer tentacles being designated "Atypical." After a week's
time the two types of regenerates cannot be distinguished as the
"atypical" hydra slowly regenerate a normal number of tentacles.
Only by cutting and observing at 48 hours can we distinguish the
typical from the atypical by gross observation. We do find differ-
ences in their interstitial cell patterns, though. These atypical sub-
clones have maintained their "atypicalness" now for two years.
A third intrinsic factor studied — the aging of asexual clones

— apparently has little effect on regenerative processes.

A fourth intrinsic factor — the effects of symbiotic relation-
ships (this term is used in the broad sense, including the relation-
ships of parasitism and mutualism) upon regeneration in hydra

— was investigated when opportunities arose on two different
occasions: (a) when the microsporidial infection of our Strain I
(H. littoralis) clones was eradicated by the fungicide Fumidil in
some of the subclones; and (b) when "non-green" clones of Chloro-
hydra viridissima were developed by culturing organisms in the
absence of light for a period of time. One of the two symbiotic
relationships studied (that in Chlorohydra) does not appear to
affect the process; the other (microsporidial infection) does affect
regeneration somewhat — the parasitized organisms regenerating
more slowly than those which have been freed from parasites.

Three types of extrinsic factors — ( a ) mechanical, ( b ) environ-
mental, and ( c ) chemical ( "foreign agents" ) — were capable of
having profound effects as measured by our gross observations.

One mechanical factor — the severance — was found to in-
fluence regeneration markedly, as the site of cutting determines to
a greater or lesser degree the subsequent response to other varia-
bles. In standardizing his analytical procedures for measuring the
regenerative response. Dr. Ham recognized the importance in sev-
ering the tentacles just below the hypostome. The importance of
cutting close to the hypostome consistently cannot be overem-
phasized. In the course of later investigations, it was noted that

ROBERT E. EAKIN 405

marked variations occurred in rate of regeneration in hydra from
a uniform subclone in the same dish of rephcates. In an effort to
trace the source of this \ ariabihty, many factors were considered
and examined. Among these was a comparison of the regenera-
tion of some hydra that were cut while in the normally contracted
state and others in the normally stretched state. It was found
that the hydra cut in the contracted state regenerate at a slower
rate than those cut while in a stretched state. To determine whether
this was due to the removal of more tissue or to the occurrence
of a larger wound in the hydra cut in the contracted state, some
of the stretched hydra were cut just behind the hypostome and
some at the mid-stomach region (midway between the hypostome
and the budding region). Since the hydra were stretched while
cut at both locations, the size of the wound was consistent but
the amount of tissue remoNcd varied. By referring to Table 1 again,
we can see the differences that could result if the cutting were
not accurately done — or if the organisms were not in a fully
extended state at the time of cutting.

The variation in regeneration rate caused by cutting the differ-
ent hydra groups at the two locations is not the same in all the
hydra tested. The ChJorohijdni and Hydra of Strain I were only
slightly retarded in regeneration rate when cut at the mid-stomach
region, whereas the regeneration rate in Strains II, III, and IV
was quite retarded. The strains of hydra whose regeneration rate
is decreased by cutting at the mid-stomach region also regenerated
fewer tentacles than control hydra cut at the hypostomal region,
a finding that can be explained by the observations made on
interstitial cell differentiation and distribution to be discussed later.

As has been reported previously (2, 4), adding a foreign chem-
ical or varying a natural environmental factor (pH, inorganic ion
concentrations, osmolarity, temperature, or the adequacy of nutri-
tion) can affect the regenerative response. In re-examining these
factors in the present study, two new observations were made:
( a ) that often the qualitative effect of a physical or chemical agent
will depend upon the exact site of severance; and (b) that, as
is shown in Table 3, the temperature at which the stock clones
have previously been maintained can affect the rate at which the
experimental organisms later will respond under the standard tem-

406

THE BIOLOGY OF HYDRA : 1961

TABLE 3

Effect of temperature on regeneration

Tentacle Numbers at 48 hours

Organisms

23-25°*

18-20°°

Strain II —Typical

5.2

6.0

Strain II —Atypical subclone No.

1.2

2.9

Strmn II —Atypical subclone No.

1.1

3.5

Strain II —Atypical subclone No.

.76

4.1

Strain III

1.2

5.1

Each value represents the average of 18 replicates.

"Maintenance temperature of stock clones; temperature during period of regeneration
was 27° for both groups.

peiature (27^) used during the established regeneration testing
procedure, a phenomenon subsequently shown to be related to differ-
ences in the interstitial cell patterns of the organisms maintained
at the two temperatures.

The effects of nutrition on regeneration rate were pointed out
by Ham (1) who found that even the elimination of one feeding
has an adverse effect upon the regeneration rate for a few days
thereafter. A more detailed study of this effect showed that: (a)
one day's fasting markedly lowered the regenerative capacity of
the organisms that were cut in the mid-stomach region, the regen-
erative ability being lowered, in fact, just as much by a 24 hour
fast as is observed after three and five day periods of fasting;
(b) but the regenerative ability of those cut just behind the hypo-
stome was not significantly aftected until the hydra had been
starved for three or more days.

In studying depressed organisms, it was found that some "slight-
ly depressed" and "moderately depressed" hydra are capable of
regenerating as fast as, or faster than, the control hydra at 18
hours, and some are able to regenerate as many tentacles. Although
not all the "depressed" and "slightly depressed" hydra regenerate
as well as the controls, the fact that some are capable of doing
so would indicate that depression in itself does not interfere signi-
ficantly with the regeneration rate. Only severely depressed hydra
are unable to regenerate. These hydra, however, are unable to
eat so that their regenerative ability may be impaired according to

ROBERT E. EAKIN 407

the extent of fasting they have undergone. Also, since these are
small contracted spherical hydra, it is very difficult to remove
the tentacles and hypostomes accurately at the desired site.

Observations on all these and other variables impressed us
even more emphatically with the fact that, before meaningful
studies can be made on the action of any type of chemical or
physical treatment "foreign" to the natural environment, one must
have used extreme care in controlling all the factors just discussed.

Also, postulates advanced concerning mechanisms controlling
the rate and extent of regeneration should offer some explanation
of these differences observed in the regenerative response.

CHEMICAL INHIBITION OF REGENERATION

The second aspect of our hydra research program which I
wish to discuss concerns the effects of chemical agents upon the
normal regenerative processes in well fed organisms. In order to
find agents which would have selective action upon the regen-
eration process without materially affecting the normal mainten-
ance and budding activities of the organism, hundreds of com-
pounds having physiological or pharmacological effects on other
types of life were screened, and from these a few compounds
having marked effects were selected for more intensive study.

From the known physiological roles of the compounds that
inhibit regeneration and alter tentacle number, the nature of a
number of the processes involved in regeneration can be implied.
In order to gain even more information concerning the mode of
action of these compounds, the organisms were exposed to them
for short intervals at different periods during the regenerative proc-
ess. It was found that a four hour exposure at the proper time was
adequate to obtain the effects of most of the compounds. We also
found that different compounds acted at different times ( Fig. 1 ) .

The results of most of these studies were published several
years ago along with a discussion of possible mechanisms involved
(2). Most of these studies were completed before we realized the
extent to which the factors just discussed were influencing the
regenerative response. Another drawback, realized at the time

408

THE BIOLOGY OF HYDRA : 1961

these screening tests were made, was the fact that we were unable
to determine the extent to which the agents being tested were
actually absorbed into the hydra cells.

TENTACLE NUMBER
ALTERATION

DLCREASL

(5-Eromuracil)

INCREASE

(Lithium chloride)

INHIBITION OF
REGENERATION

Lithium chloride

Chloretone

5 " Bromurac il

"2 Thienyl alanine

TIME IN HOURS
Fig. 1. Periods of sensitivity of hydro to regeneration-altering agents

The micro-injection technique perfected by Dr. Claybrook now
makes it possible for us to introduce these inhibitors, metabolites,
and drugs into the enteron in semi-solid agar where they will
certainly be absorbed to a much greater degree than they would be
from the environmental culture fluid. We are currently re-examining
the effects of these agents with the refinements in our experi-
mental techniques that we have developed since the initial chemi-
cal studies were made.

One of the most unique effects disclosed by the study of chemi-
cal agents was that encountered when one of the more recently
discovered members of the B vitamin complex — lipoic acid —

ROBERT E. EAKIN 409

was tested. A summary of a study we reported in detail several
years ago ( 3 ) states :

1. Hydra treated with 10 'M lipoic acid for short periods im-
mediately after removal of their hypostomes and tentacles com-
pletely lose the capacity to regenerate those structures;

2. Removal of the non-regenerating tip of such blocked hydra
leads to relatively normal regeneration;

3. The blockage of regeneration was found to be reversed in
some cases by the action of agents known to interfere with normal
nerve activity in more highly developed organisms.

At that time we postulated that the counteracting effect upon
lipoic acid inhibition by certain agents known to depress neural
activity in higher organisms was the result of interference with
some nerve-mediated reaction which was a vital part of the overall
regenerative process. This postulate seemed plausible because of
the known involvement of nerves in regeneration of amphibian
limbs. It was at this point that Dr. Spangenberg began her histo-
logical studies on hydra. In order to get a clear picture of the
nerve pattern in untreated and lipoic acid treated hydra, she had
to do considerable work in refining the methods then available
for staining nerve cells (8). Her intensive and exhaustive studies
failed to reveal any observable differences in the nerve cells of
regenerating and inhibited organisms. However, her efforts were
certainly not wasted because the study not only gave additional
information concerning the epidermal nerve net in hydra, but
disclosed that it was other type cells that were affected by the
lipoic acid treatment. ( Some of Dr. Spangenberg's histological and
cytological investigations on the normal and abnormal regeneration
of hydra are given on pp. 413-423.)

ENZYMATIC STUDIES ON A REGENERATION
INHIBITOR

In order to determine whether or not specific phenomena ob-
served in hydra were of general significance, we have used other
more highly developed systems undergoing morphogenic changes
in types of experiments suggested by the results obtained with the

410 THE BIOLOGY OF HYDRA : 1961

primitive hydra. We have made the most extensive investigations
in this respect with regeneration in planaria (5). It has been most
gratifying to find that many of the phenomena we have observed
in hydra regeneration have analogous responses in the planaria.
For example, exposure of a decapitated planaria to low concen-
trations of lipoic acid during the first part of the regenerative
process results in arrest of regeneration and the development of
an acephalic organism. These planaria appear to lead normal
lives except they do not respond to the presence of food— they
literally must be led to their piece of rat liver, but once they are
on it they feed normally. Cutting again in the non-regenerating
head area will initiate normal regeneration, a situation analogous
to that observed in hydra.

Because planaria provide larger amounts of material with which
to work, we are using this organism for enzymatic studies. Because
the lipoic acid effect is so unique in regenerating hydra and
planaria (and in a number of other organisms undergoing de-
velopmental changes), we have concentrated our efforts on
determining the biochemical mechanisms which this compound
must alter to produce the unusual morphogenic effects. It was
found (a) that the presence of oxaloacetate (but not aspartate
or a-ketoglutarate ) during the exposure of regenerating systems
to lipoic acid prevented the latter from arresting regeneration and
(b) that lipoic acid inhibited certain enzymatic activities of plana-
ria homogenates and preparations from mammalian tissues. Fur-
ther investigation on enzymes related to oxalacetate metabolism
showed the DPN-dependent malic dehydrogenase to be unusually
sensitive to lipoic acid and to other related cyclic disulfides but
not to the reduced (dithiol) derivatives. On the basis of several
types of observations on a number of other enzymatic systems,
we have concluded that the mechanism of action of these cyclic
disulfides is unique for the DPN-malic dehydrogenases ( 6 ) .

This was true not only in extracts prepared from planaria but
was shown to be the case in highly refined porcine preparations
obtained commercially. Subsequent tests with extracts of acetone
powders prepared from hydra showed that their malic dehydro-
genase activity was likewise inhibited by very dilute concentra-
tions of lipoic acid and its homologs. In both planaria and hydra

ROBERT E. EAKIN 411

preparations the relatixe activity of enzymatic inhibition of a
series of homologs strikingly paralleled their specific activity in
arresting regeneration. On the basis of these results, we proposed
that the primary action which eventually results in the inhibition
of normal morphogenesis is the inhibition of this specific enzyme.

CONCLUSION

By the use of different strains subjected to various physical and
chemical treatments one can produce a \ ariety of different regen-
eration patterns that can be recognized by macroscopic observa-
tions. Treated and untreated organisms exhibiting these different
responses can then be used for making comparative histological
studies to determine the structural differences in the processes
taking place in the different patterns of normal and altered regen-
eration. In these same organisms cytochemical, enzymatic, and
related types of biochemical studies can likewise be used for com-
parative studies. The use of a particular agent, lipoic acid, is a
beginning in this approach to establish correlation between the
effect of an agent on gross development, its effect upon cellular
patterns, and its effect upon specific biochemical reactions.

REFERENCES

1. Ham, R. G. 1957. Biochemical studies on regeneration of Inclra. Doctoral Dis-

sertation, The University of Texas.

2. Ham, R. G., and R. E.. Eakin. 1958. Time sequence of certain physiological

events during regeneration in hydra. /. Exp. Zool. 139: 33-54.

3. Ham, R. G., and R. E. Eakln. 1958. Loss of regenerative capacity in hydra

treated with lipoic acid. /. Exp. Zool. 139: 55-68.

4. Ham, R. G., D. C. Fitzgerald, Jr., and R. E. Eakin. 1956. Effects of lithium

ion on regeneration of hydra in a chemicalh- defined environment. /. Exp.
Zool. 133: 559-572.

5. Henderson, R. F., and R. E. Eakin. 1959. Alteration of regeneration in

planaria treated with hpoic acid. /. Exp. Zool. 141: 175-190.

6. Henderson, R. F., and R. E. Eakin. 1960. Inhibition of malic dehydrogenase

by cyclic disulfides. Biochem. and Biophys. Res. Comin. 3: 169-172.

7. Spangenberg, D. B., and D. L. Claybrook. 1961. Infection of hydra by Micro-

sporidia. /. Protozoology 8: 151-152.

8. Spangenberg, D. B., and R. G. Ham. 1960. The epidermal nen'c net of hydra.

;. Exp. Zool. 143: 195-202.

412 THE BIOLOGY OF HYDRA : 1961

DISCUSSION

EAKIN: When the use of oxaloacetate as a source of COo was
mentioned, during the previous discussion, the question came to
my mind as to whether or not increased CO^ tension would
reverse the inhibition of regeneration caused by hpoic acid. I
would be very chagrined if we found out that the effect of oxaloace-
tate was merely to build up the CO2 level.

BURNETT: Were yoiu- normal hydra budding when you cut them
through the middle?

EAKIN: Yes, we always use hydra that had one (or preferably
two) buds in order to insure that our organisms were in an optimal
state of nutrition. The use of the words "mid-stomach cut" was
to indicate a severance in the region mid-way between the hypos-
tome and the point of budding.

Perhaps I should explain how the differential counts on the
developing interstitial cells were made. These counts were made
on longitudinal sections through the center of the coelom. The
ratio of interstitial cells (at the four stages of maturity) to total
cells is a maximum at the hypostome and decreases as one pro-
gresses proximally until a minimum occurs just before reaching
the budding area. Past this point the ratio increases markedly,
the final stages of maturation into cnidoblasts being especially prom-
inent. The counts were made in the area between the site of sever-
ance and the line demarcating the minimum concentration of
interstitial cells, thus excluding the budding area.

A Study of Normal and

Abnormal Regeneration

of Hydra'

Dorothy B. Spangenberg

Spcin^enherg Lcihoraiorics, Refugio, Texas

One of the most challenging fields of developmental research
is the study of the regeneration of lost parts in lower animals.
This area of investigation is of special interest because most of
the processes involved in such regenerative phenomena (cell mi-
tosis, cell differentiation, cell migration, the interaction of cells or
tissues) are analagous to those taking place in most other forms of
development, and because these processes in lower animals can
be studied with relative ease.

Hydra are particularly suited for this purpose since they are
able to regenerate lost parts rapidly and can be easily maintained
in the laboratory. Previous investigators of hydra regeneration
have placed a major emphasis on (a) the capacity of different
hydra parts to regenerate, (b) histological changes during normal
regeneration, (c) regulation following abnormal regeneration, and
(d) metabolic gradients in regenerating hydra. Recent investiga-
tions made in this laboratory of the regenerative ability of hydra
(as measured by the rate of regeneration of tentacles and the
number of tentacles regenerated) revealed that normally this
capacity varies even between strains of the same species of
hydra and can be markedly influenced by environmental factors

^ These investigations were carried out at the Clayton Foundation Biochemical In-
stitute, The University of Texas, Austin, Texas.

413

414 THE BIOLOGY OF HYDRA : 1961

such as the temperature at which the hydra are tested, the
temperature at which they are maintained prior to severance, the
site at which they are cut, their state of nutrition, and intracellular
parasitism (4, 11).

A unique effect upon the regeneration of hypostomes and
tentacles was demonstrated by Ham and Eakin (5) using lipoic
acid, a cyclic disulfide which functions normally as a coenzymatic
unit in the decarboxylation of «-keto acids. By exposing "decapi-
tated" hydra to extremely dilute concentrations of this compound
they were able to "permanently" inhibit regeneration of severed
structures in Htjcha lifforolis. However, the "vitamin" was not
otherwise toxic to the organisms, and such inhibited hydra re-
generated normally if the non-regenerated tip was cut away after
several days. Subsequent studies showed that alterations in the
normal pattern of development induced by exposure to lipoic acid
were not related to the compound's enzymatic functions but rather
to a specific effect of the cyclic disulfide structure of the oxidized
form of this vitamin. In Chlorohijdra exposed to lipoic acid, the
regeneration of tentacles and hypostomes is likewise retarded early
in the regeneration period, l)ut in this species abnormal regenera-
tion subsequently occurs wherein large numbers of tentacles and
extreme body deformity results ( 13 ) . With the latter, higher
amounts were needed.

Early studies on the action of chemical agents which counter-
acted the lipoic acid effect in H. littoralis (5) indicated that the
disulfide might be affecting the nervous system of hydra. However,
cytological studies of the nervous system of normally regenerating
and inhibited hydra failed to reveal morphological evidence that
the nervous system was altered in these treated hydra. Therefore,
detailed studies of other processes either known to be or suspected
to be involved in normal regeneration were carried out using
both normal and lipoic acid treated hydra. The study of these
processes ( interstitial cell differentiation, cell mitosis, cell migration
and the interaction of cell layers) led to the development of
postulates concerning mechanisms involved in normal regenera-
tion and to postulates explaining, at least in part, the effect of
lipoic acid on hydra regeneration.

DOROTHY B. SPANGENBERG 415

INTERSTITIAL CELL DIFFERENTIATION

The role of interstitial cells in hydra regeneration has been
emphasized by many in\ estigators (1, 7, 9, 10). While the ability
of interstitial cells to differentiate into any other cell type in hydra
is disputed, most authors agree that cnidoblasts arise from inter-
stitial cell differentiation (2, 10, 14). The differentiation of inter-
stitial cells into cnidoblasts (specifically those containing desmo-
nemes) was selected as a process deserving study, since it can be
directly related to the rate of normal and abnormal regeneration
in hydra. Cell counts of differentiated stages of interstitial cells
in the pre-tentacle area of non-regenerating hydra (severed and
then immediately killed ) revealed that those hydra whose tentacles
and hypostomes had been removed close to the hypostomal area
(while the hydra were in a stretched condition) contained more
desmonemes and late-state interstitial cells than did hydra which
had been cut in the mid-stomach region. This and other observa-
tions indicated that along the body of a hydra there is a qualita-
tive gradient in the distribution of the various stages of interstitial
cells, with the greatest concentration of desmonemes and late-stage
interstitial cells at the base of the tentacles (11). Tardent (14),
using Chlorohijdra, reports a quantitative gradient in the distribu-
tion of all types of interstitial cells. The existence of a qualitative
gradient explains the decreased regenerative ability observed in
normal hydra severed at the mid-stomach region when compared
to those severed at the hypostomal region, since in the former
case most of the reserve of partially differentiated interstitial cells
is removed at the time of the cutting. It is postulated, therefore,
that any factor w4iich is apt to interfere with normal differentiation
of interstitial cells is a more effective inhibitor in hydra cut at the
mid-stomach region than in those cut at the hypostomal region.

Some of these factors which have been observed to affect in-
terstitial cell differentiation are a depletion of nutrients (fasting of
the animal prior to testing ) , and the presence of intracellular para-
sites in hydra (11). Also, the inhibition produced by exposure
to lipoic acid and to certain other chemical agents is much more
effective in hydra cut in the mid-stomach region than in those cut
at the hypostomal region.

416 THE BIOLOGY OF HYDRA : 1961

Histological studies of lipoic acid treated hydra {H. littoralis
and Chlorohydra mridissima) revealed that, at 20 hours after
cutting, many undifferentiated interstitial cells were in the pre-
tentacle area, whereas only a few late-stage interstitial cells and
cnidoblasts containing desmonemes were present. This indicates
that normal differentiation of interstitial cells does not take place
in these hydra. Morphologically, however, the interstitial cells
appear normal as compared to controls. It is questionable, there-
fore, whether the differentiation of the interstitial cells is inhibited
by a direct action of lipoic acid on these cells or whether it is in-
fluenced by damage to another cell type which may normally
contribute some substance to interstitial cell differentiation. Histo-
logical sections of lipoic acid treated H. littoralis and C. mridissima
reveal that some of the gastrodermal cells are damaged. Whether
or not this damage of gastrodermal cells is related to reduced
interstitial cell differentiation in the epidermis is still to be deter-
mined. It is believed that many factors which influence the re-
generative process do so by interfering with interstitial cell differ-
entiation.

CELL MIGRATION

The importance of cell migration in morphogenesis has been
emphasized both in embryonic development ( 16 ) and in the re-
generative process ( 15) .

Grafting segments of Chlorohydra from a normal clone (con-
taining green algae in their gastrodermal cells) to segments of a
modified clone ( no visible green algae ) revealed that during normal
regeneration gastrodennal cells migrate from closely adjacent re-
gions to participate in the regenerative reconstruction. Migration
of gastrodermal cells was not impaired in lipoic acid treated Chloro-
hydra, and in fact, the extent of cell migration appeared to be
increased (Fig. 1). It is possible that the increased migration
of gastrodermal cells following lipoic acid treatment results from
the need for replacement of the gastrodermal cells that are damaged
by the chemical ( 13 ) .

On the basis of many observations made of regeneration of

DOROTHY B. SPANGENBERG

417

Fig. 1. Migration of gastrodermal cells in grafted Chlorohydra treated
with lipoic acid.

new tentacles in H. littoralis, it is our opinion that some cnido-
blasts migrate short distances to become incorporated into the
new tentacle; however, most of the nematocysts for the new ten-
tacles are formed after cutting from the differentiation of interstitial
cells during the early part of the regeneration period. Histological
sections of lipoic acid treated Hydra offered the best evidence of
interstitial cell migration. In these organisms, where maturation
of the interstitial cells is inhibited, large numbers of undifferent-
iated interstitial cells were seen at the pre-tentacle site twenty
hours after cutting, whereas only a few cnidoblasts containing

418 THE BIOLOGY OF HYDRA : 1961

desmonemes were present. Although it was not possible to observe
migration of the immature interstitial cells, their presence in such
large numbers indicated that they had migrated. On the other hand,
there was no such accumulation of desmoneme-containing cnido-
blasts; hence, there could have been no significant amount of
migration of these cells.

CELL MITOSIS

Cell mitosis during regeneration was studied by early investi-
gators, many of whom did not regard this process as being im-
mediately essential for tentacle formation. Rowley (10), while
studying C. vindissima, concluded that the new cells are not formed
at the cut surface alone and that the tentacles do not seem to
be regenerated solely from new tissue. She felt that the new cells
which appear during the regeneration of hydra are formed by
division of the old cells throughout "the entire piece" (as in the
normally growing animal) and that the tentacles are formed from
old cells and from cells that have arisen by division of the already
differentiated cells of the old part.

Except for a slight increase in mitosis noted by Ham and Eakin
(5), reaching a maximum at 45 minutes near the site of the cut
(a reaction possibly associated with wound healing), we have not
observed any increase in mitotic activity during the remaining
regeneration period. It is believed, therefore, that mitotic activity
if diffuse throughout the body of the hydra and there is no increase
in activity during normal regeneration or in hydra treated with
lipoic acid. The presence of all stages of mitotic activity in lipoic
acid treated hydra indicated that this process had not been altered
by exposure to this disulfide.

Since interstitial cells are believed to be replaced through mitosis,
there most likely is an increase in this activity after the regeneration
period in order to replace those cells which differentiated into
the necessary components of the new tentacles. Further research,
however, will be necessary to determine the extent to which this
occurs.

DOROTHY B. SPANGENBERG 419

INTERACTION OF CELL LAYERS

The two cellular layers of hydra, the epidermis and gastroder-
mis are separated by a non-cellular substance, the mesogloea,
which varies in thickness throughout the column of the hydra.
The mesogloeal layer is extremely thin in normal hydra at the
tentacle base and at the site of a newly forming bud. In regenerating
hydra, the mesogloea is not replaced at the regenerating tip until
the early tentacles have appeared and the hypostome has formed.
In lipoic acid treated Chlorohijdra, areas which are lumpy in form
show a decrease or absence of mesogloea ( 13 ) ; and according to
Chang, Hsieh, and Liu (3), in depressed hydra, prior to develop-
ment of abnormal form, the mesogloea "dissolves." In all of
these instances, in areas where there is active differentiation of
interstitial cells prior to normal or abnormal growth of tissue, the
mesogloea appears reduced or absent. Conversely, at the tentacle
bases of fasted hydra and in the stalk region of normal hydra,
where there is little interstitial cell differentiation taking place, the
mesogloea appears to be relatively thick. In several hydra which
have been permanently inhibited by lipoic acid, at 48 hours after
cutting, the mesogloea has been restored although the hydra have
not regenerated.

The foregoing does not imply that interstitial cell differentia-
tion takes place only in areas where there is no visible mesogloea.
A certain amount of interstitital cell differentiation is undoubtedly
taking place continually in areas that contain visible mesogloea.
However, we believe that the differentiation process is hastened
in areas of active growth by a closer contract of the two tissue
layers in the absence of visible mesogloea.

Whether the mesogloeal material is actually utilized in areas of
active cellular differentiation is not known. If the mesogloea does
contain collagen or a collagen-like material as postulated (12),
then it is possible that this substance may be utilized in a manner
analagous to its use in wound healing in higher animals. Whether
or not such use is made remains to be determined.

Where the mesogloea is not seen in various areas of the hydra,
however, the gastrodermis and epidermis appear to be in closer

420 THE BIOLOGY OF HYDRA : 1961

contact. Gastrodermis removal and fasting studies have indicated
that a normal amount of gastrodermis and proper nutrition are
necessary for normal regeneration. It is possible that a transfer of
typical nutrients, or possibly some other specific substance neces-
sary for epidermal interstitial cell differentiation, might be better
facilitated when the two cellular layers are in closer contact. If
this is the case, then variations in the amount of mesogloea might
be an important factor in growth regulation of hydra by in-
fluencing the extent of interstitial cell differentiation.

Induction of the differentiation of one tissue by its close contact
with another tissue has been studied extensively, but the exact
mechanism is not known. Although it is interesting to compare
the situation in hydra (wherein close contact of gastrodermis and
epidermis occurs during active interstitial cell differentiation) to
induction in embryological studies, there is no direct evidence that
the mechanism is the same. However, Moore (8), working with
the hydroid Coidijloplwrci lacustris, noted that oral cone grafts
will induce from mass tissue the development of hydranth regions
basal to the oral cone. She observed that no induction was pro-
duced when an oral cone graft was separated from the host tissue
by an agar or a cigarette paper barrier. "Direct close contact
between the graft and the host appears to be necessary for induc-
tion to be produced."

The origin of mesogloeal material during regeneration is not
known. In regenerating hydra, the mesogloea is restored in the
hypostomal region, usually by 18 hours regeneration time, after
interstitial cell differentiation has occurred. That the mechanisms
involved in restoration of the mesogloea are not identical with
those involved in interstitial cell differentiation is apparent in
lipoic acid treated Hydra littoralis, wherein interstitial cell
differentiation has not occurred normally by 18 hours, yet the
mesogloea is restored at the same rate as in normally regenerating
controls. If one considers that a normal balance must occur be-
tween the rate of interstitial cell differentiation and mesogloeal
restoration, then a growth regulating mechanism can be postulated
wherein alteration of either of these processes can cause a variation
in the normal regeneration pattern.

DOROTHY B. SPANGENBERG 421

RETARDED INTERSTITIAL CELL DIFFERENTIATION
WITH NORMAL MESOGLOEAL RESTORATION

111 normal regeneration, this relationship could account for
variation in the regenerative ability of normal hydra. It has been
demonstrated that the normal reserve of partially differentiated
interstitial cells varies in different strains of hydra (11). The time
required for the acquisition of the necessary number of differentiat-
ed interstitial cells is dependent upon the quantity of reserve differ-
entiated cells already available. Variation in amount of such re-
serves and in the rate of differentiation producing new ones could
account for \'ariation in normal regenerative ability of hydra pro-
vided the mesogloeal material is restored in all hydra at the
same rate.

In lipoic acid "inhibited regeneration" {H. littoralis), the dif-
ferentiation of interstitial cells is definitely retarded, yet the meso-
gloeal material is restored at a normal rate. Once the mesogloeal
material is restored, it appears that the interstitial cells do not
differentiate sufficiently for regeneration to occur. However, if the
non-regenerated tip (and consequently the mesogloeal layer) is
removed several days hence (after cellular damage has been re-
paired) normal regeneration occurs.

RETARDED INTERSTITIAL CELL DIFFERENTIATION
WITH RETARDED MESOGLOEAL RESTORATION

This condition is observed in lipoic acid treated Chlorohydra
where the inhibitory effect expresses itself in one of two ways —
(a) either permanent inhibition (as in H. littoralis) or (b)
retardation with subsequent "wild" growth ( 13 ) . In case ( a ) , the
restoration of the mesogloeal mechanism may occur before suffi-
cient interstitial cell differentiation has occurred (as in lipoic acid
treated H. littoralis), and tlie hydra would be permanently in-
hibited. In case (b), the restoration of the interstitial cell differ-
entiation may occur prior to the restoration of the mesogloea,
resulting in the differentiation of more interstitial cells than nor-
mally occurs, and the hydra exhibit the "wild" growths observed.

422 THE BIOLOGY OF HYDRA : 1961

These postulated mechanisms are very speculative, but they do
offer a basis for planning future investigations concerned with
the mechanisms of interstitial cell differentiation and of mesogloeal
restoration during regeneration and the interplay of these two
factors as a growth controlling mechanism in regeneration.

CONCLUSIONS

Many factors (both intrinsic and extrinsic) influence the differ-
entiation of interstitial cells to cnidoblasts during regeneration.

Other cell types must be considered as possible contributors
to the process of interstitial cell differentiation.

The mesogloea is not visible in areas where intense interstitial
cell differentiation is proceeding in normal hydra or severed hydra
( both in untreated and lipoic acid treated organisms ) .

A growth regulating mechanism is proposed wherein a balance
between the quantity of mesogloea present in an area of the
hydra and the extent of cell differentiation (apparently brought
about by the close contact of the gastrodermis and the epidermis)
must be achieved for normal regeneration to occur.

Although the emphasis has been placed upon the importance
of cell migration, cell differentiation and the interaction of cell
layers during the regenerative process, other mechanisms (many of
which are still unknown) must also contribute to this very complex
process.

A knowledge of the interactions occurring throughout the whole
animal, especially the interrelationship l^etween difterent cells and
cell layers through chemical and physical interchange, must be
acquired before a true understanding of the overall process of
regeneration will be achieved.

REFERENCES

1. Brien, p. and M. Reniers-Decoen, 1949, La croissance, la blastogencse, et

I'ovogenese chez YHijdra fusca (Pallas). Bull. biol. France et Belg. 83:
293-386.

2. Burnett, A. L. 1959. Histophysiology of growth in hydra. /. £.v/). Zool. 140:

281-342.

DOROTHY B. SPANGENBERG 423

3. Chang, J. T., H. H. Hsieh, and D. D. Liu. 1952. Observations on hydra, with

special reference to abnormal forms and bnd formation. F/n/.s-. Zool. 25:
1-10.

4. Ham, R. G., D. C. Fitzgerald, and R. E. Eakin. 1956. Effects of litliium ion

on regeneration of hvdra in a chemically defined environment. /. Exp.
Zool. 133: 559-572.

5. Ham, R. G., and R. E. Eakin. 1958. Loss of regenerative capacity in hydra

treated with lipoic acid. J. Exp. Zool. 139: 55-68.

6. Ham, R. G. and R. E. Eakin. 1958. Time sequence of certain physiological

events during regeneration in Hydra. J. Exp. Zool. 139: 33-54.

7. Kanajew, /. 1930. Zur Frage der Bedeutung der interstiticllen Zellen bei Hvdra.

Roux. Arch. 122: 736-759.

8. Moore, J. 1951. Induction of regeneration in the hydroid Corchilophora lacustris.

J. Exp. Biol. 28: 72-93.

9. Moore, J. 1952. Interstitial cells in the regeneration of Cordt/lopJiora lacustris.

Quart. J. Microsc. Sci. 93: 269-288.

10. Rowley, H. 1902. Histological changes in Hydra viridis during regeneration.

Amer. Naturalist 36: 578-583.

11. Spangenberg, D. B., and R. E. Eakin. 1961. A study of variation in the regen-

eration capacity of hydra. (Manuscript submitted)."

12. Spangenberg, D. B., and R. E. Eakin. 1961. Histological studies of mechan-

isms involved in hydra regeneration. ( Manuscript submitted ) . *"

13. Spangenberg, D. B. and R. E. Eakin. 1961. The effect of lipoic acid on regen-

eration of Chlorohydra viridissima. (Manuscript submitted).*

14. Tardent, p. 1954. Axiale Verteilungs-Gradienten der Interstiticllen Zellen bei

Hydra und Tuhidaria und ihre Bedeutung fur die Regeneration. Roux
Archiv. 146: 593-649.

15. Tardent, P. 1960. Developing Cell Systems and TJieir Control. 18th Growth

Symposium, The Ronald Press Company, New York.

16. Wagner, R. P., and H. K. Mitchell. 1955. Genetics and Metabolism. John

Wiley and Sons, Inc., New York.

"The investigations reported in these three manuscripts submitted to the Journal
of Experimental Zoology have been published as a doctoral dissertation: A Study of
Mechanisms Involved in Normal and Abnormal Regeneration of Hydra, 1960. The
University of Texas.

Growth Factors
in the Tissues of Hydra

Allison L. Burnett^

Universite libre de Bnixcllcs, Bnixelles, Belgium, and
The University of Virginia, Charlottesville, Virginia

THE METABOLIC GRADIENT OF HYDRA

The work of Child and Hyman (7) demonstrated that hydra
possesses body regions which show a striking difference in met-
abohc activity. This finding was corroborated by Hinrichs (9),
Weimer (20, 21, 22) and Child (5, 6). These investigations re-
vealed in general that hydra possesses a primary apico-basal
gradient with a secondary increase in metabolic activity in the
budding region. The areas of the tentacles and peduncle were
shown to have a low metabolic activity as compared to the hypo-
stome and budding region, and the basal disk, while less active
than the hypostome, was found to possess a metabolic activity
higher than that of the gastric region and much higher than that
of the tentacles and peduncle. Actually, it is unwise to state that
hydra possesses a gradient at all, for it isn't a gradient in the true
sense of the word. Hydra simply has three very active regions,
the hypostome, budding region, and basal disk; one fairly active
region, the gastric region; two regions of low metabolic activity,
the peduncle and tentacles.

It is interesting to speculate on the factors which account for
these differences in metabolic activity along the length of the body
column. Burnett (3) has conducted a nutritional study on hydra
during periods of rich feeding and prolonged starvation. He found

^Present address: Department of Biology, Western Reserve University, Cleveland,
Ohio.

425

426 THE BIOLOGY OF HYDRA : 1961

that the concentration of food reserves ( glycogen, neutral fats,
protein reserve droplets) along the body column is directly in
proportion to the metabolic activity of a particular region. Areas
of low metabolic activity contain few gastrodermal inclusions while
areas which are capable of reducing methylene blue when applied
to the living animal contain an excess of food reserves. Further-
more, a histological examination of different body regions in
hydra has revealed that interstitial cells and gland cells are scarce
or lacking altogether in regions of low metabolic activity and are
abundant in regions of high activity.

A question which immediately comes to mind is reminiscent
of the old "hen and egg" question, i.e. do regions of high metabolic
activity possess this activity because of the presence of food in-
clusions and interstitial cells in this area, or do these areas contain
these specific cells and food inclusions because of the general
active metabolism of this area which is not directly related either
to interstitial cells or specific food inclusions?

Nearly all workers in the field of metabolic gradient have
stressed the fact that the "head" region (hypostome region), be-
cause of its high metabolic activity, in some manner supresses the
formation of another head in its immediate vicinity. For this
reason, a bud never forms directly beneath the head of the parent
under normal conditions, but begins as an outpushing of the body
roughly midway between the hypostome and basal disk. Such a
hypothesis immediately suggests that the "head" region liberates
an inhibitive substance which in some manner prevents the cells
of the adjacent gastric region from entering the active cell divisions
which would eventually lead to the formation of a bud. If this is
indeed true, then it is first necessary to demonstrate that such an
inhibitive principle exists, and secondly, if it does exist, it is
necessary to determine why it doesn't affect cellular divisions in
the head region itself.

Another series of questions are closely linked with this same
problem. Brien ( 1 ) has shown that the sub-hypostomal region
of hydra is an active growth center. Constant cell proliferation
in this center forces cells distally towards the tentacle tips and
proximally towards the basal disk where these "migrating" cells
atrophy and are sloughed off the body column. Thus, by constant

ALLISON L. BURNETT 427

cell proliferation in the hypostomal region and constant cell death
at the extremities, hydra is able to grow continually yet maintain
its form.

Numerous grafting experiments conducted by several different
investigators, notably Rand (15, 16, 17) Hefferman (8), Browne
(2), Kolitz (11), Burt (4), Issajew (10), Rand, Bovard, and
Minnich (18), Tripp (19), Mutz (14), Yao (23, 24, 25), demon-
strated that the hypostomal region of hydra is the "dominant"
center of the animal and will induce polyp formation at the site
where it is grafted to the body column of another hydra. Similarly,
it was shown by many of these workers that tissues of the develop-
ing bud will induce hydranth formation in another hydra at the
graft site.

From the obser\ations cited thus far in this paper, it would
appear that the hypostomal region of the animal is one which is
engaged in constant growth activities. In this highly metabolically
active region a growth inhibitive principle is produced which
passes down the stalk and inhibits cellular dixisions in the adja-
cent gastric region. In the budding region of the hydra, the in-
hibitive principle presumably does not exert its affect and the cells
in this area take on properties similar to those in the hypostomal
growth region. In a sense, it may be stated that a new growth
region is created in the budding region, for both the hypostomal
and budding regions engage in cell divisions which lead to the
formation of a new polyp. If this is correct, then it must be assumed
that cells in the gastric region possess the potential of growth
and rapid cell division, but that these cells are inhibited in some
manner from performing these vital functions while they are lo-
cated in the gastric region.

This paper will attempt to answer the following basic questions.
What accounts for the high metabolic activity of a particular area?
Does hydra possess a specific growth inhibitive principle which
is produced in the hypostome? Does this principle become inac-
tivated as it diffuses down the body column towards the budding
region? Does this principle inhibit cellular divisions in the area
in whch it is produced? Are there specific substances responsible
for the growth and inductive potential of the hypostome?

None of the experiments designed to answer these basic ques-

428 THE BIOLOGY OF HYDRA : 1961

tions will be described in detail in this paper. A long monograph
describing the growth processes exhibited by hydra is now in
press (/. Exp. ZooL). This monograph will describe in detail all
of the experiments listed in the present paper.

THE GROWTH INHIBITING PRINCIPLE IN HYDRA

In order to demonstrate that hydra possesses a growth inhibit-
ing principle in its tissues, and that this principle is produced in the
hypostomal region, a simple experiment was conducted. It was as-
sumed that if an inhibitive principle was produced in the hypo-
stomal region and that if this principle diffused in some manner
down the body column, it would be possible to induce head forma-
tion in the gastric region of the animal by simply preventing the
flow of this principle. Such a manuever was accomplished by sim-
ply grafting the peduncle of one hydra between the hypostomal
and gastric region of another animal. Burnett (3) has shown that
the peduncular digestive cells are highly vacuolated and have a
wasted, aged appearance. It was thought that perhaps these vacu-
olated cells might in some way impede the passage of a growth in-
hibitive principle proximally.

Thirty-nine grafts similar to that described in the preceding para-
graph were performed. In 19 cases the hypostomal region plus the
transplanted peduncle split from the gastric region and the original
peduncle. After the split the gastric region grew a new "head" on
its distal surface. However, in 20 cases, after a period of 1-2 days,
tentacles formed in the gastric region just below the site of the
transplanted peduncle. Eventually an entire hypostome was
formed proximal to the grafted peduncle, and the outcome of this
experiment was the formation of two completely normal hydra.

This experiment clearly indicates that cells in the gastric region,
when removed from the direct influence of the hypostomal region,
are stimulated to form a new "head" region. It may be possible
that a growth inhibitive substance produced in the hypostomal
region diffuses down the body column inhibiting substances which
would normally promote growth in the gastric cells. Moreover, the
inhibitive principle must pass down the column by diffusion from

ALLISON L. BURNETT 429

cell to cell since there was a direct connection between the head and
gastric regions through the gastrovascular cavity.

Another series of experiments were undertaken to determine
why the inhibitive principle is not effective in the budding region
of the animal, and whether the inhibitive principle affects the divid-
ing cells in the hypostomal region where it is produced.

It appeared possible that as the inhibitive principle diffused
proximally along the gastric region that it might become more
dilute or perhaps inactivated or broken down after it remained for a
given period of time in the gastric region. If this hypothesis is cor-
rect, then it should be possible to suppress the asexual reproductive
process by placing the growth region closer to the budding region
than it is normally.

It was shown that if the gastric region of the hydra is removed,
and the growth region grafted to the area adjacent to the budding
region, budding will not occur until growth process in the hypo-
stomal region have forced the "head" some distance away from the
budding region. This distance is roughly porportional to the area
occupied by the excised gastric region. However, it was also shown
that if the head is transferred to a site adjacent to a budding region
which has already begun bud formation, the bud goes on to form
normally and is not inhibited by the transplanted head.

These experiments suggest that once a region is actively in-
volved in growth processes it is not influenced by the growth in-
hibitive principle. This observation explains why the inhibitive
principle is not effective in the area in which it is produced. It
exerts its effect only on those cells which have been pushed proxi-
mally because of growth processes in the hypostomal region. How-
ever, these experiments also indicate that budding can be sup-
pressed by the presence of a hypostomal region in the immediate
vicinity if cell division which would eventually lead to the formation
of a bud has not begun.

In view of these results it appears that once active cell divi-
sion in the budding region begins this region is similar to the hy-
postomal growth region of the parent. Neither of these two regions
are influenced by the inhibitive principle; both are, in a sense, form-
ing an entirely new individual. Perhaps this analogy can be further
extended, and it may be hypothesized tliat the developing bud

430 THE BIOLOGY OF HYDRA : 1961

liberates an inhibitive principle similar to that liberated from the
head region of the parent.

Lenlioff ( 12 ) has demonstrated that, if the gastric region of a
H. littoralis containing a bud is transected immediately above that
bud, head formation of the parent is inhibited at the site of section
even after the bud separates from the parent stalk. This observation
strongly indicates that an inhibitive substance is released from the
tissues of the bud. However, the answer is not as simple as that.
If it is true that the inhibitive substance l^ecomes more dilute as it
diffuses along the column, the regeneration of the parent head
should not be affected if the head is transected at the level of the
sub-hypostomal growth region rather than through the gastric
region as demonstrated by Lenhoff.

In order to test this hypothesis, parent hydra, bearing buds
in all stages of development were transected either through the
middle of the gastric region or through the sub-hypostomal growth
region. Subsequent examination of these animals revealed that ani-
mals which had been excised through the growth region regener-
ated within twenty-four hours and were not affected by the presence
of a bud on their column. However, animals which had been ex-
cised through the gastric region were inhibited in their regenerative
processes and had not even begun tentacle formation after twenty-
four hours. Interestingly enough, parent animals, excised through
the gastric region and containing buds which had formed peduncles
had begun tentacle formation after twenty-four hours. It will be re-
membered that the peduncular region is capable of inhibiting in
some manner the passage of a growth inhibiting principle.

These results indicate that a developing bud does contain a
growth inhibiting principle, and that this principle in some way
is gradually rendered impotent as it diffuses from the bud along
the length of the parent column.

Many aspects of hydra's biology can be tentatively explained
in light of a growth inhibitive substance. For instance, many specu-
lations have been made to explain why a hydra containing several
buds always produces them successively, and why the buds, in ad-
dition to the fact that they are in different stages of development,
are arranged in a helical pattern and are on essentially different
sides of the stalk from one another.

ALLISON L. BURNETT 431

The budding pattern may operate through the following mech-
anism. When a bud first begins its development an inhibitive prin-
ciple is released. This principle does not permit the formation of
another bud in adjacent regions of the parent column. As the bud
continues to grow it forms tentacles and eventually a peduncle; at
this time a second bud begins to form on the opposite side of thr
parent stalk. Presumably, the peduncle inhibits the flow of the in-
hibitive principle back to the parent stalk, also the principle
which has previously diffused back into the parent before peduncle
formation, is more concentrated on the side of the stalk adjacent
to the bud than on the opposite side. The third bud will form on the
opposite side of the stalk and above the second bud. Again, the
formation of the third bud does not occur until much of the energ>^
supply of the second bud in the form of food reserves is depleted,
and the second bud has begun tentacle and peduncle formation.
Such a mechanism makes it virtually impossible for two buds to
compete for food materials from the same area of the parent col-
umn. However, the author has observed that under conditions of
extremely rich feeding, it is not unusual for two buds to begin to
form simultaneously from the same level of the parent stalk and di-
rectly opposite one another. Since both buds have begun to form
simultaneously, their inhibitive principles will have no effect upon
one another.

Furthermore, the presence of an inhibitive principle explains
why cells which pass from the budding region down into the
jDeduncle have a low metabolic activity, are highly vacuolated,
and contain few food reserves. First, most of the food reserves which
were originally in these cells have been utilized during bud for-
mation. Secondly, subsequent ingestion of food by the hydra will
not nourish the peduncular cells because they are under the di-
rect growth inhibiting action of the neighboring budding region and
do not require large amounts of food for the upkeep of this metabol-
ically inactive region. If the peduncle is excised from the body
column it will never completely regenerate into a normal hydra
because of a lack of energy reserves. An excised peduncle is capa-
ble of forming only 2 or 3 tentacles when excised from the inhibiting
action of the budding region.

432 THE BIOLOGY OF HYDRA : 1961

GROWTH STIMULATING PRINCIPLE IN HYDRA

It is interesting at this time to consider the factors which stimu-
late growth in hydra and which are under the direct control in
certain body regions of growth inhibiting principles. In order to
demonstrate the existence of a growth principle in hydra it is first
necessary to define the action of this principle. It has been previ-
ously stated that hydra possesses two "growth" regions in its body,
the hypostomal growth region and the budding region. It is well
known that if the hypostome is removed from a hydra, a new hy-
XDOstome is always formed on the distal portion of the excised body
column. The formation of a new hypostome may be interpreted as
follows: after the excision of the hypostome, the gastric region is
no longer under the influence of the growth inhibiting principle
which normally diffuses proximally from the hypostomal region.
Therefore, growth substances present in the gastric region are
activated and a new growth center is established. On the other
hand, if an animal is excised through the gastric region, the
proximal portion of the region containing the hypostome al-
ways forms a new gastric region, peduncle, and base — never an-
other hypostomal region. Again we may say that a growth inhibit-
ing principle from the hypostome is inhibiting head formation in the
proximal region.

If the foregoing analysis is correct, it should be possible to
initiate head formation in the proximal portion of an excised gas-
tric region by supplying additional amounts of the growth principle
to this area. Presumably an excess of a growth stimulating princi-
ple would overcome the influence of the growth inhibiting prin-
ciple.

In order to demonstrate that a growth principle exists in meta-
bolically active regions of the hydra, and that this principle is
capable of diffusing from these regions and stimulating cell growth
and division in adjacent regions, the following experiments were
conducted.

Two different species of hydra were employed in these experi-
ments. One species was the common brown Pelmatohydra oligactis;
the other was a new species, Htjdra pirardi, recently discovered in
Belgium by Dr. Paul Brien. When these two species are grafted to

ALLISON L. BURNETT 433

one another there is no celhilar exchange whatsoever between the
species, except that the nematocysts of one species are able to be
incorporated into the tissues of the other species.

Twenty Pelmatohydra oligactis were excised through the mid-
dle of the gastric region, and the distal excised portions which con-
tained the head region were grafted to the growth regions of a sim-
ilar number of Hydra pirardi whose hypostomes and tentacles had
been excised. After a period of 2-3 days the distal regions of the
H. pirardi portions began to form new tentacles and hypostomes.
The following day, tentacle growth invariably began on the proxi-
mal region of the H. oligactis portions. Ultimately, new head
regions were formed on either side of the junction of the grafted por-
tions and the grafted animals separated from one another. A true
reversal of polarity had thus been effected in P. oligactis, and this
had been accomplished without any exchange of cellular material
from H. pirardi with the possible exception of cnidoblast cells which
would presumably not be directly involved in growth processes.

These results indicate that a growth stimulating principle is pres-
ent in the hypostomal region of H. pirardi and that this principle is
capable of passing into the tissues of Pelmatohydra oligactis and
stimulating head formation.

A further series of experiments were conducted to further con-
firm the presence of the potential of the growth stimulating princi-
ple. It will be recalled that cells of the peduncular region of hydra
are highly vacuolated, contain few food inclusions, are metabolically
inactive, and are destined to die and be sloughed off the basal
disk. Furthermore, the epithelio-muscular cells of his region con-
tain very little cytoplasmic RNA, and this area is characterized
by the fact that it contains few or no interstitial cells and no glan-
dular cells in its proximal regions.

Thus, it was desirable to determine whether a growth stimulat-
ing principle, after being introduced into the peduncular region,
would be capable of "rejuvenating" this senescent region.

Fifty peduncular regions of Pelmatohydra oligactis were grafted
to the growth regions of a similar number of Hydra pirardi as in
the previous experiment. In 47 cases the peduncles of the P. oligactis
portions formed a basal disk and separated from the H. pirardi
portions before the latter had begun head formation. However, in

434 THE BIOLOGY OF HYDRA : 1961

3 striking instances H. pirardi portions began tentacle formation
before the H. oligactis portions had detached. In these 3 cases
the results were most interesting. Small tentacles began to form
from the peduncles of F. oligactis a day after tentacle formation.
had begun on H. pirardi. When the grafts were fed with brine
shrimp, it was noticed that the bulk of ingested food materials were
taken into the digestive cells of the peduncle of F. oligactis. Such a
phenomenon never occurs under normal conditions.

Three days after feeding the 3 animals were sectioned for his-
tological study. It was found that the normally wasted peduncular
digestive cells of F. oligactis were full of protein reserve droplets.
Interstitial cells had invaded this area and appeared in concentra-
tions comparable to the normal growth region of the hydra. Sev-
eral dozen interstitial cells had transformed into gland and mucous
cells, and in the lower regions of the peduncle where a basal disk
would normally be expected to form, a new hypostome was nearly
completely elaborated.

Thus, it appears that F. oligactis does contain specific growth
stimulating principles within its tissues. When these principles (or
principle) are present in a body region in sufficient concentrations,
this region will take in large amounts of food after each feeding and
will subsequently be invaded by interstitial cells. It is hypothesized
that the metabolic activity of a given region of hydra is dependent
upon the amount of growth stimulating principle which is present
and which is not being affected l)y a growth inhiliiting factor.

Unfortunately little is known at the present time concerning the
nature of either the growth stimulating or inhibitive principle. A
method has recently been devised whereby it is possible to collect
the stimulating principle in agar blocks and introduce it into any
desired body region of another hydra. Burnett and Schwager are in
the process of elucidating the chemical nature of this principle.

The growth inhibiting principle has not been isolated from the
tissues of the hydra at the present time. It will be interesting to de-
termine whether the inhibitive principle which acts in the tissues
of the hydra by diffusing proximally from the growth region is the
same as that found by Lenhoff and Loomis (13) which can limit
the asexual reproductive process of a colony of H. littoralis. The
inhibitive principle described by Lenhoff and Loomis is heat-

ALLISON L. BURNETT 435

labile, dialyzable, non-gaseous, and absorbed on the cation-exchange
reagent permutit. They have extracted this principle from the cul-
ture medium in which H. lifforoUs have been crowded.

REFERENCES

1. Brien, p., and M. Reniers-Decoen, 1949. La croissance, la blastogenese,

I'ovogenese chez Hydra fusca (Pallas). Bull. Biol. France et Belg., 82:
293-386.

2. Browne, E. 1909. The production of new hydranths in hydra by the insertion

of small grafts. }. Exp. Zool. 7: 1-23.

3. Burnett, A. 1959. Histophvsiologv of growtli in hydra. /. Exp. Zool. 140:

281-342.

4. Burt, D. R. 1925. The head and foot of Pclmatohydra oligactis as unipotent

systems. Arch, fiir Entwick. mech. 104: 421-433.

5. Child, C. M. 1934. Differential reduction of methylene blue by li\ing organisms.

Proc. Soc. Exp. Biol, and Med. 32: 34-36.

6. Child, C. M. 1947. Oxidation and reduction of indicators by hvdra. /. Exp.

Zool. 104: 154-195.

7. Child, C. M., and L. H. Hyman, 1919. Axial gradients in the hvdrazoa. I. Biol.

Bull. 36: 183-221.

8. Hefferman, M. 1901. Experiments in grafting hvdra. Arch, fiir Entwick. mech.

13: 567-587.

9. Hinrichs, M. N. 1924. A demonstration of axial gradient by means of photolysis.

/. Exp. Zool. 41: 21-32.

10. Issajew, W. 1925. Studien an organischen Regnlationen ( Experimentelle Unter-

suchungen an Hydren). Arch, fiir Entwick. mech. 108: 1-67.

11. Koelitz, W. 1911. Morphologische and experimentelle Untersuchungen an

Hydra. Arch, fiir EiUwick. mech. 31: 423-455.

12. Lenhoff, H. 1957. The induction of a new center of polarity in regenerating

Hijdra littoralis. Anat. Rec. 127: 325.

13. Lenhoff, H., and Loomis W., 1957. The control of clonal growth of Hydra

by the self inhibition of tentacle differentiation. Anat. Rcc. 127: 429.

14. MuTZ, E. 1930. Transplantationsyersuche an Hydra mit besonderer Berucksich-

tigung der Induction, Regionalitat, und Polaritiit. Rotix' Arch. fiir. Entwick.
mech. 121: 210-271.

15. Rand, H. W. 1899. The regulation of graft abnormalities in h>dra. Arch, fiir

Entwick. mech. 9: 161-214.

16. Rand, H. W. 1899. Regeneration and Regulation in Hydra viridis. Ibid. 8: 1-34.

17. Rand, H. W. 1911. The problem of fonn in Hydra. Science 33: 391.

18. Rand, H. W., Bovard, J. F., and D. E. Minnich, 1926. Location of fonnation

agencies in hydra. Proc. Nat. Acad. Sci. U.S.A. 12: 565-570.

19. Tripp, K. 1928. Regenerationsfahigkeit yon Hydren in den yerschiedenen Kor-

perregionen nach Regenerations und Transplantationsversuchen. Zschr.
Wiss. Zool. (Korshelt Festband), 132: 476-525.

20. Weimer, B. R. 1928. The physiological gradient of hvdra. Physiol. Zool. 1:

183-230.

21. Weimer, B. R. 1932. The physiological gradient of hydra. /. Exp. Zool. 62:

93-107.

436 THE BIOLOGY OF HYDRA : 1961

22. Weimer, B. R. 1934. The physiological gradient of hydra. Pliysiol. Zool. 7:

212-225.

23. Yao, T. 1945. Studies on the organizer problem in P. oligactis. J. Exp. Biol. 21:

147-150.

24. Yao, T. 1945. Effect of some respiratory inhibitors and stimulants and of

oxygen deficiency on the induction potency of the hypostome. /. Exp.
Biol. 21: 150-155.

25. Yao, T. 1945. Bud induction by developing hypostome. /. Exp. Biol. 21: 155-

160.

DISCUSSION

STREHLER: How was it determined that cells move up into
the tentacles and die at the tips?

BURNETT: Semal-Van Gansen (1951) vitally stained limited
areas of the tentacles and watched the stain migrate distally.

STREHLER: Do both the gastrodermal and ectrodermal layers
move towards the tip of the tentacle?

BURNETT: Yes, both move. In 1926 Issajew observed that if a
fork forms in the tentacle of a hydra, the fork will move distally
becoming progressively smaller until it gradually disappears at
the tentacle tip. We have observed this many times. Such a phenom-
enon would not occur unless both cell layers were migrating. More-
over, cnidoblasts are steadily pushed into the tentacles through such
growth processes from the sub-hypostomal growth region.

STREHLER: Then the cnidoblasts don't migrate as free cells?

BURNETT: In Pelmatohydra oligactis, there are always free
nematocysts in the enteron and in the digestive cells of the tentacles.
This can be demonstrated in the following experiments. The proxi-
mal portion of a methylene blue stained animal is grafted to the
distal portion of an unstained animal. Under conditions of normal
feeding, stained nematocysts are not found in the epidermal
batteries, but only in the gastrodermal cells of the tentacles. How-
ever, if the nematocyst supply in the tentacle batteries is depleted,
then stained nematocysts are transferred from the digestive cells of
the tentacle to the epidermal batteries. It is impossible for stained
nematocysts to reach the tentacle through growth processes because
the stained nematocysts are all proximal to the growth region, and

ALLISON L. BURNETT 437

growth would serve only to push the nematocysts towards the base
of the animal.

CROWELL: If you cut the tentacles off, nematocysts first form
in the column, and then move into the tentacles from the lower
region. How do they get there?

BURNETT: They are simply forced distally by an active cell
proliferation in the growth region of the hypostome.

CROWELL: No, I'm not talking about the ones that grow nor-
mally, but when one cuts the tentacles off and mobilizes nematocyst
formation elsewhere, how do these cnidoblasts move?

BURNETT: The answer is still the same. Cnidoblasts of a newly
regenerated tentacle are pushed there by sub-hypostomal growth
processes. Some cnidoblasts are pushed into the peduncle. Others
pass into the enteron where they are swept to the tentacles;
many are ingested by digesti\e cells in this area. However, in
Pelmatohyclm oligactis these ingested cnidoblasts will be trans-
ferred to an adjacent tentacle battery only if that battery's sup-
ply of nematocysts is depleted.

CROWELL: They are transported in the gastro-vascular cavity?

BURNETT: That's right.

LOOMIS: There is another piece of evidence suggesting that nem-
atocysts reach the tentacles through the gastro-vascular cavity. This
is the well-known fact that when the worm Micwstomiim eats
hydra, the undischarged nematocysts migrate through the worm's
tissues until they are in position in the ectoderm and ready to be
used by him. In other words, if nematocysts can travel through the
endoderm to the ectoderm of a flatworm, then almost certainly they
can do the same through the tissues of a hydra. This would explain
how a hydra can arm its tentacles within twenty four hours after
being fed e\'en though it takes about a week for new cells to grow
out from the hypostome to the end of a tentacle.

BURNETT: Also, I believe that the nematocysts in the worm
Microstomum are moved passively through the tissues of the worm.
I think the nematocyst is phagocytized by a worm mesodermal cell

438 THE BIOLOGY OF HYDRA : 1961

and is transported to the epidermis. I do not think that the hydra
cnidoblast is active in the process.

FULTON: What do you find in the gastrovascular cavity? Are
these nematocysts or cnidoblasts?

BURNETT: In the worm the nematocyst is naked, as I understand
it. In the hydra the nematocyst is always inside a cnidobkist.

SLAUTTERBACK: We have sought an answer to that question
with the electron microscope and have never seen a nematocyst
in the tentacles of hydra which was not still within the cnidoblast
which produced it. It seems fair to assume that if the nematocyst mi-
grates it does so within its own cnidoblast.

Also we have seen a cnidol^last migrating through the mesoglea
only once. It seems rather unlikely to me that very many cnidoblasts
can pass through the mesoglea and be repeatedly missed by us.
We have studied H. littoralis, P. oligactis and C. viridissima.

BURNETT: This is interesting. H. pirordi appears to have a cnido-
blast migration pathway different from tliat of P. oligactis. I have
examined Dr. Brien's slides of H. pirardi and have observed that the
digestive cells in the base of the tentacle contain many ingested
nematocysts. Some of these nematocysts, in section, can even be
seen traversing the mesoglea towards the tentacle battery.

SLAUTTERBACK: When nematocysts are seen in the gastro-
derm one must be very careful to determine that they are not de-
generating and that they are still within a living cnidoblast.
Many of these may appear to be variable even when examined
quite critically with the light microscope but at higher magnifica-
tions there are often signs of cytoplasmic degeneration in the cnido-
blasts and deterioration of the nematocysts themselves.

BURNETT: Yes, we have made the same observation. However,
many of the cnidoblasts plus the enclosed nematocyst in the di-
gestive cells of the gastrodermis are normal. We have shown that
when a proximal portion of P. oligactis is grafted to the distal region
of H. pirardi, nematocysts characteristic of H. oligactis pass into the
enteron of H. pirardi where they are ingested. If this ingestion takes
place in the tentacles, the nematocysts of P. oligactis are passed

ALLISON L. BURNETT 439

on to the epidermal batteries of H. pirardi where they are still cap-
able of discharging.

CROWELL: I'm sure cnidoblasts have to migrate and I'm pretty
sure we're not yet clear on how they do it. Now another aspect of
the same thing. You spoke of inducing interstitial cells in the basal
region. Were these cells there, or did they move in?

BURNETT: The interstitial cells migrated from more distal re-
gions of the animal. I am almost certain of that.

CROWELL: Did they invade by way of the gastrovascular tract,
or did they creep? What happens when they get there?

BURNETT: I don't think interstitial cells ever migrate via the
gastrovascular cavity. Brien has studied interstitial cell migration
from a small portion of normal hydra grafted to a hydra whose
interstitial cells were killed through X-irradiation. He observed an
epidermal migration of interstitial cells. We have noted a similar
phenomenon in our induction experiments.

CROWELL: Were they creeping? Are they wriggling between
epidermal cells?

BURNETT: I've never seen them creep. I suppose they migrate
in an amoeboid fashion. I have never seen interstitial cells in the
gastrodermis at any stage during the induction phenomenon.

CROWELL: I think Nelson Spratt has seen migrating cells work-
ing their way along. Were they interstitial cells?

FULTON: No, they were nematoblasts, l^ut Dr. Spratt made
his observations on Tubiilaria.

BURNETT: I'm not sure that undifferentiated cells are capable
of migrating. I am referring to the small basophilic cells seen in
nests along the length of the epidermis. Each time that I observe a
"migrating cell" it is much larger than the cells found in these epi-
dermal nests. Perhaps a "migrating cell" is a partially differenti-
ated interstitial cell.

FAWCETT: It seems to me that these cells can only migrate at
either end of this differentiation sequence. They could migrate

440 THE BIOLOGY OF HYDRA : 1961

as an individual interstitial cell or after the cluster has completed its
differentiation and separates again into individual cells. It would be
quite impossible for a syncytial cluster of 8 or 16 cells connected
by these bridges, which I believe in very firmly, to insinuate them-
selves between other cells in the column or to cross the mesoglea
and get into the gastrovascular cavity and re-invade at a higher
level. I think this syncytial relationship almost excludes any migra-
tion in the interim period. They will either have to migrate as un-
differentiated individual interstitial cells, or as cnidoblasts that have
matured nematocysts within them.

STREHLER: I wonder whether you've ever seen migration of
any nematocysts or cnidoblasts into the tentacles of Campanularia.
In this case they could not move through the gastrovascular cavity
because the tentacles have no cavity. Also, I would like to ask why
you call that pigment lipofuscin.

BURNETT: The inclusions found in Hydra pirardi fed on Ar-
temia are not carotenoids and are not dissolved by lipid solvents.
They are dense bodies, often found in clusters, and as I remember,
they stain only after they have been oxidized by permangenate or
a similar oxidizing agent,

STREHLER: If it was not extractable with organic solvents, then
perhaps you should call it "hydrafuscin." What were those slides
stained with?

BURNETT: Methylene blue.

STREHLER: Was that the natural color of those granules?

BURNETT: Yes, the methylene blue didn't go into the granules.

STREHLER: Do you find them down toward the base?

BURNETT: Yes, like the carotenoids in hydra, they are especial-
ly concentrated in the hypostome, budding region and basal disk.
They probably represent some type of excretory crystal. They per-
sist for a greater length of time during starvation. I am not quali-
fied to comment any further on their nature or function.

Nucleic Acid

and Protein Changes

in Budding Hydra Uttoralis

Yu-YiNG Fu Li

AND

Howard M. Lenhoff

Laboratories of Biochemistnj, Howard Hughes Medical Insiitute, and Departments
of Biochemistnj and Zoology, University of Miami, Miami, Florida

Budding in hydra has excited biologists since it was first discov-
ered over 250 years ago by Leeuwenhoek and Trembley. As one step
toward understanding the mechanisms involved in the initiation
and growth of a l^ud, we have been searching for means to charac-
terize chemical differences between parent and bud tissues (2).
Some of our preliminary observations concern changes in the amount
and distribution of DNA, RNA, and protein in budding Hydra.

Experiments involving the chemical analyses of cellular compo-
nents required large number of Hydro in the same stages of bud-
ding. These were obtained by carefully controlling the time at
which they were fed, while keeping all other growth conditions
constant. In these experiments, Hydra having one bud were removed
from a mass culture (3). The animals were then allowed to starve
4-6 days, during which time the original bud and one or two latent
buds completed their development, detached, and were discarded.
On the sixth day of starvation the animals were fed once with excess
Artemia nauplii. As shown in Figure 1, nearly all of the Hydra
initiated a small bud within the first day after feeding.

The developmental stages of the budding Hydra are presented
in Figure 2. First, the 6-day starved animal enters the "small bud"

441

442

THE BIOLOGY OF HYDRA : 1961

X
1-

DC
Q

>
X

Li_

o
cr

LU
GO

8 -

r—

p-o-

■o-o-o-o-o

—

/
/

■6

—

/
/
/

—

/
/

oo-o-o-o-o-o

—

FEED

1 I

1 1 1 1 1

1 1

1 1 1 1 1 !

6 -

4 -

6 8

DAYS

Fig. 1. Time of bud initiation in starved Hydra after one feeding. This
is a representative experiment on 5 Hydra. Similar results were obtained
using hundreds of Hydra.

stage 12 hours after one meal. Once the bud is initiated, it contin-
ues its development through the "medium bud" stage, the "short
tentacled bud" stage, and the "long tentacled bud" stage. The gas-
trovascular cavity of each bud in the "long tentacled" stage are no
longer connected to the parent's cavity, and the buds are now
capable of detaching. After its detachment the new bud is then fed
until it in turn begins to initiate its own bud. Throughout this entire
process we followed the chemical changes occurring in two major
phases: bud development initiated by one feeding, and the growth
and development of the detached bud to the adult stage.

The protein, DNA, and RNA values of a six day starved Hydra
following a single feeding and during the ensuing stages of bud-
ding are shown in Figure 3. It should be emphasized that all DNA

LI AND LENHOFF

443

"short tentocled
bud" stage

"long tentocled
bud" stage

feed to

maturity

Fig. 2. Stages of budding Hydra.

and RNA measurements were made without separating these macro-
molecules from the rest of the cellular components. Thus, the DNA
and RNA values also include the smaller species of these molecules.
The DNA, RNA, and protein were assayed, using the diphenyla-
mine (1), orcinol (5), and Lowry (4) methods respectively. The
protein, DNA, and RNA values for Hydra before feeding (not
shown on this figure ) were about half that of their respective values
at 12 hours. Thus, on feeding, a doubling in all of these components
occurred because of the ingested protein, DNA, and RNA of the
shrimp. During the next 72 hours, however, these macromolecular
components decreased slowly, while the DNA protein ratio re-
mained relatively constant. The changes observed probably resulted
from at least two factors: (a) degradation of the ingested food, and
(b) the synthesis of Hydra cellular components.

In Figure 4 are shown the DNA protein ratio of Hydra in the
long-tentacled bud stage and that of the same animals 48 and 72
hours later when the buds have detached from the parent. (The
protein content of the bud was about one third that of the parent. )

444

THE BIOLOGY OF HYDRA : 1961

10
SB

20 30
MB

40 50 60
ST

HOURS AFTER FEEDING

Fig. 3. DNA, RNA, and protein content of Hydra in different stages of
budding. The symbols SB, MB, ST, and LT represent the small bud, medium
bud, short tentacled, and long tentacled stage animals.

These data reveal the first major chemical difference between par-
ent and bud tissues, the DNA/ protein ratio of the bud being three
times that of the parent Hydra.

Since the experiments in Figure 3 gave no indication of the bud
possessing this high DNA/protein ratio, another type of experiment
was carried out to determine whether this high ratio was already
present in an early stage of bud tissue. In these experiments (Fig.
5 ) , we excised only the bud portion of a "medium bud" stage Hydra
and then determined the respective chemical composition of the
dissected parents and buds. It can be seen that, although there is
much less protein in the dissected bud than in the remaining parent,
the DNA contents of both parts are nearly equal. More striking is
the high DNA/protein ratio of the dissected bud. These results

LI AND LENHOFF

445

0.6 f-

0.5

± 0.4

LlI

o
q:

^ 0.3
<

0.2 -

72 120 144

LT P B P B

HOURS AFTER FEEDING

Fig. 4. DNA protein ratio of long tentacled stage Hydra (LT) before and
after the buds detached. P and B represent parents and buds. Ten animals
are used in each experiment.

indicate that a high DNA protein ratio is a property of bud tissue in
its early development. It is also possible, although less likely, that
the high DNA values in bud tissues actually represent large pools
of diphenylamine-positive material which serve as precursors for
DNA synthesis.

The chemical changes occurring in buds fed until they reach

446

THE BIOLOGY OF HYDRA : 1961

32 -

28 -

>
o

O
<

D/Pr.

Pr.

D/Pr.

Pr.
VI R

DISSECTED
PARENT

DISSECTED
BUD

- 1.0 - 160

^ _i

H.9
.8
- .7
-.6?

H.4 <

H.3

.2
H .1

- 40

- 20

-" 0

140

120 <
or

100 ^
o

80 =L

60 fe
a:

Q-

Fig. 5. DNA, RNA, and protein content of buds dissected from 10 medium
bud stage Hydra, and of the parents remaining after dissection.

parent size is shown in Figure 6. The first sets of measurements
were made before the animals were fed. The last measurement was
made two days after the third feeding. The results show that the pro-
tein and RNA increased to that of the small bud stage Hydro. In
contrast, the DNA, which was high initially, decreased slightly.
Consequently, the DNA/protein ratio decreased until it approached
that of the parent Hydra in the small bud stage.

The consistent finding of this study was the relatively high
DNA/protein ratio of buds (Figs. 4, 5, 6). Consequent with growth,

LI AND LENHOFF

447

-1 120

Isf feeding 2nd feeding 3rd feeding

DAYS BUDS ARE GROWING

100

80
60
40

en
20 Q.

Fig. 6. DNA, RNA, and protein content of buds during growth to small
bud stage Hydra.

owing to an increase in protein, the bud's DNA/protein ratio dimin-
ished to that of the parent ( Fig. 6 ) . These experiments suggest that
in bud cells the amount of nuclear material is high relative to the
cytoplasm, and that subsequent growth of the detached bud
involves an increase in cytoplasmic components rather than mitosis.

REFERENCES

1. Burton, K. 1956. A study of the conditions and meclianism of diphenylamine

reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem.
}. 62: 315-323.

2. Li, Y.-Y. F. and H. M. Lenhoff. 1960. Nucleic acid patterns of Hydra budding

in synchrony. Anat. Rec. 137: 376.

3. LooMis, W. F. and H. M. Lenhoff. 1956. Growth and sexual differentiation of

hydra in mass culture. /. Exp. Zool. 132: 555-574.

4. LowRY, O. H., N. J. RosEBROUGH, A. L. Farr and R. J. Randall. 1951. Protein

measurement with the Folin phenol reagent. /. Biol. Chem. 193: 265-275.

5. Schneider, W. C. 1957. Determination of nucleic acids in tissues by pentose

analysis. Methods in Enzymologij 3: 680-691. Academic Press, Inc., New
York.

448 THE BIOLOGY OF HYDRA : 1961

DISCUSSION

BURNETT: I might mention that if the end of a bud is excised
early in its development, the remaining part will regenerate while
still attached to the tissues of the parent. This suggests that cellular
divisions are occurring within the tissues of the bud proper. More-
over, we have been able to demonstrate that after the bud hypo-
stome reaches a certain distance from the parent column, it develops
a growth region of its own. The tentacles of the bud arise only when
the tip of the bud has grown some distance from the parent. Tenta-
cle formation is dependent upon cellular divisions in the budding
tissue itself, and tentacular material is not furnished by the parent.

CLAYBROOK: I'd like to report an observation about which we
have no further information. Occasionally, in cultures of Hydra lit-
toralis growing at a minimal rate while being fed the heated Artemia
diet which I reported on yesterday, we find buds that fail to form
hypostomal tentacles or mouth. They may remain attached to the
parent for days or weeks. Sometimes they detach after they have
differentiated a basal disk, but some still do not produce anv ten-
tacles. Under these conditions, they cannot eat and eventually
disintegrate.

Index

Acid phosphatase,

aginji, increases on, 385

of Ciimpanularia, 381, 386

of ectoderm, 385

of j^astrodermis, 385

of hydra, 381, 384, 385

of hsosomes, 381

of pedal disk, 384, 385

of tentacles, 384

of tentacles, Campamdaria, 386
Acontia, 179, 180, 184, 190

5-h\dro.\ytryptamine of, 180, 184

nematocysts of, 190
Acrasin, 375
Acrifla\ine,

Artcmia, labeled with, 388

CampanularuL labeled with, 388
Acrophore, 189
Acropora cenicornis, calcification in, 272,

285
Acropora luilntata. 285
Action potentials,

of CorclyIo)>lior(i, 293

of h>dra, 75, 76

of Tulnilaria, 293
Activators, of feeding reflex, 210
Adenosine triphosphate, of aging Cam-

paniilaria, 389
Aging, 373

acid phosphatase, increase in, 385

adenosine triphosphate of Campami-
laria during. 389

of Campaniihria, 302, 303, 375, 379,
387-391

on cellular replacement, 393

of Ccreiis peduncithitus, 374, 375

in coelenterates, 373-398

Aging,

of corals, 396

defined, 373

of human myocardium, 378, 379

of himian skin, 376

of hydra, 373, 379

of Maniciiui arcolata, 395

models for, 376

of Ohdia commissiirulis, 375

radiation on, 390, 391

on regeneration, 404

theory of, 393, 394
Ahermatvpic corals,

defined, 270

distribution of, 270

growth properties of, 270
Albino Clilorohijdra, see C. viridissima,

albino.
Algae,

calcareous, 270

in calcification, 285

in reef building, 279

see cdso S>'mbiotic algae

see also Zooxanthellae
Alkaline phosphatase, of corals, 284
Amastigophore, 189
Amino acids,

on feeding reflex, 211

of Pliysalia toxin, 173
Anmionia gas,

amoiuit in sexual cultures, 349

in dense culture of hydra, 350

micromethod for, 345

permeabilit\' of cell membrane to, 349,
358

on pH of culture solution, 349, 350

toxicity, varies with pH, 349

449

450

INDEX

Ammonium bicarbonate,

presence in halo zone, 349

released by hydra, 349, 361
Ammonium ion, impermeability of cell

membrane to, 349
Amoeboid motion, factors affecting, 357
Ampliirna,

calcification by, 276

carbon fixation by, 276
Anacrophore, 189
Anaplotelic, 189
Anatomical regions, of hydra, 1
Anions, on feeding reflex, 213, 215
Anisorhizic, 189
Anthopleiira elegant issima,

algae, role of, 258

algae, absence of, 255

animal to algae ratio in, 266

symbiosis in, 255

tissue culture of cells, 245-254

toxins of, 177

weight changes on star\ation, 258

zooanthellae of, 255-257
Arthropods, in reef building, 270
Asexual reproduction,

in Craspedacusta, 318

in hydra, 441-448
Asparthione, on feeding reflex, 211
Astomocnidae, defined, 188
Athecate hydroids,

growth zones of, 298

hydranth development, 298

reconstitution of, 305
Atoll, 269
Attachments, hydra, 51-66

between epithelial cells, 62

between epithelial cells and mesoglea,
62
Atrich, 189

discharge of, 194

role of, 194
Aurelia, strobilization of, 336

Bicarbonate,

on pH of culture solution, 350
pH, relation to, 361
source of, for calcification, 274, 275
Bioassay, for hydra growth factor, 235
Biological variability, 399
BouganvilUa, longevity of, 311
Branch tube, 292
Brom cresol purple, visualization of mi-

croenvironment with, 338
Bud, hydra,

chemical changes during growth of,

446
developmental stages of, 441, 443
DNA/protein ratio of, 445, 446
growth of, 354
histology of, general, 8
initiation of, 441, 442
initiation of, in synchrony, 441
jimction with parent column, 8, 42
origin of, 426
Budding,

Cia.s))ed(iciista, 3 17-336
factors influencing, 318
feeding rates on, 329
interactions in, 323, 331
pattern of, basic, 320
patterns of, 317, 320-323, 327
physiological interactions of, 320
sequence of, 320, 321

nutridon on, 320, 325-328, 330,

331, 335
temperature on, 318, 32-325, 330-

334
temperature optima, 320
hydra, 441, 446

effect of sexual difterentiation on,

366
inhibition of, 448
chemical changes during, 441-446
Buds, Craspedacusta, types of, 319
Bufotenin, 167, 186
Butyrobetaine, 180

Banthine, 181

Basal disk, general histology, 7

Basal granule, 85, 86

Basal processes, of epithefia, 51

Basitrich, 189

Battery, of Physalia nematocysts, 169

Bicarbonate, 353

deposition, assay of, 275

equilibrium with pH and pCO^, 361

pCOj, relation to, 361

Calcification, 269-285

in Acropora cervicornis, 272

by ahermatypic corals, 275-277

assay of, 271

carbon dioxide, role of, 282-284

carbonic anhydrase on, 272

by corals, 269

Diamox on, 272

diurnal rates of, 277

factors affecting, 271

by hermatypic algae, 275-277

INDEX

451

Calcification,

by herniatypic corals, 275-277

light, role of, 279

mechanism of, 273, 280

oxaloacetate on, 283

oxygen on, 273

photosynthesis, role of, 272, 277, 278,

280
rates of, 275, 278
removal of waste on, 274
species differences in, 272
stimulation of, 273
theory of, 273
zooxanthellae, role of, 270, 279, 283,

285
Calcium carbonate,

deposition, 271, 273, 280

assay of, 275

photosynthesis on, 271
production,

mechanism of, 273

in reefs, 270, 273
Calcium ions,

for calcification, source of, 274, 275
on feeding reflex, 214, 225
CaUiactis parasitica, 184
behavior of, 198

function of nematocysts in, 198-202
5-hydroxytryptamine of, 184
Calyptoblast, 297
Campaniihiria,

acid phosphatase of, 381, 386
acid phosphatase, of tentacles, 386
aging of, 302, 375, 379, 388, 391
cell migration in, 440
culture of, 302
desmosome of, 66
development of, 291-316, 375, 376
food catching ability of, 388
growth pattern, alterations of, 301
histochemical properties, 379
hydranth,
' ATP, content of, 389

differentiation of, 299, 307

ingestion time by, 388

regression of, 304, 379, 380-383

replacement of, 304
longevity of, 311
mitosis in, 313
nutrition on, 301
patterns of colonial growth of, 298-

300, 311
peristalsis of, 379
radiation on, 312, 390, 391, 395
reconstitution of, 304-306

Campanidaria,

regeneration of, 308, 309, 397

regenerative capacity of, 307

regression of, 304, 379-383

section of, 381

temperature on, 301

tentacle of, 387

terminal growth, age on, 303

transport in, 397
Campanularia ficxtio.sa, 297, 300, 331

see Campanularia
Capitulum, of Craspedacusta polvp, 317,

319
Capsule, nematocyst, 81, 112, 132, 154,

155
Carbon dioxide, 410

calcification, role in, 283, 284

loss to environment, 362

incorporated by hydra cells, 362

as a metabolite, 361, 362

pemieates cell membrane, 358

in replacement of dicarboxvllic acids,
362

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